Systems biology of the modified branched Entner-Doudoroff pathway in Sulfolobus solfataricus.

Sulfolobus solfataricus is a thermoacidophilic Archaeon that thrives in terrestrial hot springs (solfatares) with optimal growth at 80°C and pH 2-4. It catabolizes specific carbon sources, such as D-glucose, to pyruvate via the modified Entner-Doudoroff (ED) pathway. This pathway has two parallel branches, the semi-phosphorylative and the non-phosphorylative. However, the strategy of S.solfataricus to endure in such an extreme environment in terms of robustness and adaptation is not yet completely understood. Here, we present the first dynamic mathematical model of the ED pathway parameterized with quantitative experimental data. These data consist of enzyme activities of the branched pathway at 70°C and 80°C and of metabolomics data at the same temperatures for the wild type and for a metabolic engineered knockout of the semi-phosphorylative branch. We use the validated model to address two questions: 1. Is this system more robust to perturbations at its optimal growth temperature? 2. Is the ED robust to deletion and perturbations? We employed a systems biology approach to answer these questions and to gain further knowledge on the emergent properties of this biological system. Specifically, we applied deterministic and stochastic approaches to study the sensitivity and robustness of the system, respectively. The mathematical model we present here, shows that: 1. Steady state metabolite concentrations of the ED pathway are consistently more robust to stochastic internal perturbations at 80°C than at 70°C; 2. These metabolite concentrations are highly robust when faced with the knockout of either branch. Connected with this observation, these two branches show different properties at the level of metabolite production and flux control. These new results reveal how enzyme kinetics and metabolomics synergizes with mathematical modelling to unveil new systemic properties of the ED pathway in S.solfataricus in terms of its adaptation and robustness.

Unraveling the function of the two Entner-Doudoroff branches in the thermoacidophilic Crenarchaeon Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 is a thermoacidophilic archaeon that metabolizes glucose and galactose via an unusual branched Entner-Doudoroff (ED) pathway, which is characterized by a non-phosphorylative (np) and a semi-phosphorylative (sp) branch. However, so far the physiological significance of the two pathway branches is unknown. In order to address these questions two key enzymes of the branched ED pathway, the class II glycerate kinase (GK) of the np-ED branch and the 2-keto-3-deoxygluconate kinase (KDGK) of the sp-ED branch in S. solfataricus, were investigated. GK was recombinantly purified and characterized with respect to its kinetic properties. Mg(2+) dependent Sso-GK (glycerate + ATP → 2-phosphoglycerate + ADP) showed unusual regulatory properties, i.e. substrate inhibition and cooperativity by D-glycerate and ATP, and a substrate-inhibition model was established fitting closely to the experimental data. Furthermore, deletion of the sp-ED key enzyme KDGK in S. solfataricus PBL2025 resulted in a similar growth phenotype on glucose as substrate compared with the wild-type. In contrast, the mutant showed strongly increased concentrations of np-ED intermediates whereas the hexose and pentose phosphates as well as trehalose were decreased. Together the results indicate (a) that the np-ED pathway is able to compensate for the missing sp-ED branch in glucose catabolism, (b) that in addition to its catabolic function the sp-ED pathway has an additional although not essential role in providing sugar phosphates for anabolism/gluconeogenesis and (c) that GK, with its unusual regulatory properties, seems to play a major role in controlling the flux between the glycolytic np-ED and the glycolytic/gluconeogenetic sp-ED pathway.

"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 method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast.

Although microbial metabolome analysis has now become a widely used method, no generally applicable quenching method has been published so far. Either the methods were established for only one defined organism or the metabolite coverage was quite low. In the current work, a novel, reliable, and robust quenching method for different types of organisms is described. Compared with the commonly used quenching procedure with 60% methanol (-50 degrees C), we obtained improved results for three examined organisms with different cell wall and membrane structures using a 40% ethanol/0.8% sodium chloride solution (-20 degrees C). Increased metabolite levels were achieved for 60-80% of all identified compounds. Moreover, the estimated standard error of the relative concentrations of 120-160 different substances was only 14+/-4% compared with 17+/-3% in unquenched samples and 24+/-7% in samples quenched with methanol for the different tested organisms.

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.