Sulfolobus

We have initiated a project to sequence the 3Mbp genome of the thermoacidophilic archaebacterium Sulfolobus solfataricus P2. Cosmids were selected from a provisional set of minimally overlapping clones, subcloned in pUC18, and sequenced using a hybrid (random plus directed) strategy to give two blocks of contiguous unique sequence, respectively, 100389 and 56105bp. These two contigs contain a total of 163 open reading frames (ORFs) in 26–29 putative operons; 56 ORFs could be identified with reasonable certainty. Clusters of ORFs potentially encode proteins of glycogen biosynthesis, oxidative decarboxylation of pyruvate, ATP-dependent transport across membranes, isoprenoid biosynthesis, protein synthesis, and ribosomes. Putative promoters occur upstream of most ORFs. Thirty per cent of the predicted strong and medium-strength promoters can initiate transcription at the start codon or within 10 nucleotides upstream, indicating a process of initial mRNA-ribosome contact unlike that of most eubacterial genes. A novel termination motif is proposed to account for 15 additional terminations. The two contigs differ in densities of ORFs, insertion elements and repeated sequences; together they contain two copies of the previously reported insertion sequence ISC 1217, five additional IS elements representing four novel types, four classes of long non-IS repeated sequences, and numerous short, perfect repeats.

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-009-0290-y) contains supplementary material, which is available to authorized users.

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔHcal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.

Until recently, the only archaeon for which a bona fide origin of replication was reported was Pyrococcus abyssi, where a single origin was identified. Although several in silico analyses have suggested that some archaeal species might contain more than one origin, this has only been demonstrated recently. Two studies have shown that multiple origins of replication function in two archaeal species. One study identified two origins of replication in the archaeon Sulfolobus solfataricus, whereas a second study used a different technique to show that both S. solfataricus and Sulfolobus acidocaldarius have three functional origins. These are the first reports of archaea having multiple origins. This finding has implications for research on the mechanisms of DNA replication and evolution.

Systems biology research typically involves the integration and analysis of heterogeneous data types in order to model and predict biological processes. Researchers therefore require tools and resources to facilitate the sharing and integration of data, and for linking of data to systems biology models. There are a large number of public repositories for storing biological data of a particular type, for example transcriptomics or proteomics, and there are several model repositories. However, this silo-type storage of data and models is not conducive to systems biology investigations. Interdependencies between multiple omics datasets and between datasets and models are essential. Researchers require an environment that will allow the management and sharing of heterogeneous data and models in the context of the experiments which created them. The SEEK is a suite of tools to support the management, sharing and exploration of data and models in systems biology. The SEEK platform provide...

The methods presented here summarize phylogenetic relationships of genomes in visually appealing and informative figures. Dekapentagonal maps depict phylogenetic information for orthologous genes present in five genomes, and provide a pre-screen for putatively horizontally transferred genes. If the majority of individual gene phylogenies are unresolved, bipartition histograms provide a means of uncovering and analyzing the plurality consensus. Analyses of genomes representing five photosynthetic bacterial phyla and of the prokaryotic contributions to the eukaryotic cell illustrate the utility of the methods.

Systems biology research typically involves the integration and analysis of heterogeneous data types in order to model and predict biological processes. Researchers therefore require tools and resources to facilitate the sharing and integration of data, and for linking of data to systems biology models. There are a large number of public repositories for storing biological data of a particular type, for example transcriptomics or proteomics, and there are several model repositories. However, this silo-type storage of data and models is not conducive to systems biology investigations. Interdependencies between multiple omics datasets and between datasets and models are essential. Researchers require an environment that will allow the management and sharing of heterogeneous data and models in the context of the experiments which created them. The SEEK is a suite of tools to support the management, sharing and exploration of data and models in systems biology. The SEEK platform provide...

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a Mr of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His108, Asp109, and His168. We also identified Glu142 as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.