Melanie Zaparty

Reconstruction of the Central Carbohydrate Metabolism of Thermoproteus tenax by Use of Genomic and Biochemical Data

The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.

The complete genome sequence of Thermoproteus tenax: a physiologically versatile member of the Crenarchaeota.

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078T) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.

Hot standards for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon Thermoproteus tenax : the unidirectional TreT pathway

In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis––the unidirectional TreT pathway––and discuss its physiological role as well as its phylogenetic distribution.

SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bacteria or Eukarya, e.g. the unusual branched ED (Entner-Doudoroff) pathway, which is utilized for glucose degradation in S. solfataricus. This archaeal model organism of choice is a thermoacidophilic crenarchaeon that optimally grows at 80 degrees C (60-92 degrees C) and pH 2-4. In general, life at high temperature requires very efficient adaptation to temperature changes, which is most difficult to deal with for organisms, and it is unclear how biological networks can withstand and respond to such changes. This integrative project combines genomic, transcriptomic, proteomic and metabolomic, as well as kinetic and biochemical information. The final goal of SulfoSYS is the construction of a silicon cell model for this part of the living cell that will enable computation of the CCM network. In the present paper, we report on one of the first archaeal systems biology projects.

The central carbohydrate metabolism of the hyperthermophilic crenarchaeote Thermoproteus tenax: pathways and insights into their regulation

Although the complexity and modifications of the archaeal central carbohydrate metabolism (CCM) are well established, the knowledge about its regulation is rather limited. The facultatively heterotrophic, hyperthermophilic crenarchaeote Thermoproteus tenax utilizes a modified version of the reversible Embden-Meyerhof-Parnas (EMP) and the catabolic, branched Entner-Doudoroff (ED) pathway for glucose metabolism. Glucose is completely oxidized to carbon dioxide via the oxidative tricarboxylic acid (TCA) cycle, which is supposedly used in the reductive direction for carbon dioxide fixation under autotrophic growth conditions. Elemental sulfur is used as final electron acceptor. The CCM of T. tenax has been well studied on protein level as well as on gene level by performing a focused transcriptional analysis (CCM DNA microarray). In contrast to the classical pathways found in Bacteria and Eucarya allosteric regulation seems to play a minor role, therefore emphasizing the important role of regulation on transcript level in T. tenax. Whereas the EMP pathway and the TCA cycle show a highly coordinated regulation on gene level, the catabolic, branched ED pathway reveals no strong regulation. The CCM pathways in T. tenax and the current understanding of their regulation are presented.

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.

DNA Microarray Analysis of Central Carbohydrate Metabolism: Glycolytic/Gluconeogenic Carbon Switch in the Hyperthermophilic Crenarchaeum Thermoproteus tenax

In order to unravel the role of regulation on transcript level in central carbohydrate metabolism (CCM) of Thermoproteus tenax, a focused DNA microarray was constructed by using 85 open reading frames involved in CCM. A transcriptional analysis comparing heterotrophic growth on glucose versus autotrophic growth on CO2-H2 was performed.

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

An additional glucose dehydrogenase from Sulfolobus solfataricus: fine-tuning of sugar degradation?

Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner-Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61% amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency. In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways.