“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

Systems biology of the modified branched Entner-Doudoroff pathway in <i>Sulfolobus solfataricus</i>

<div><p><i>Sulfolobus solfataricus</i> 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 <i>S</i>.<i>solfataricus</i> 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 <i>S</i>.<i>solfataricus</i> in terms of its adaptation and robustness.</p></div

Model validation.

<p>Comparison between and <i>SS</i>^<i>70°C</i> for metabolite and flux steady states. A-B) Pie Chart. Inner circle: percentual contribution of each metabolite (A) or flux (B) steady states to the overall steady state at 70°C (<i>SS</i>^<i>70°C</i>); Outer circle: percentual contribution of each metabolite (A) or flux (B) steady states to the overall steady state at 70°C () (0% contributions ignored); C-D) Bar Chart comparing the metabolite and flux steady states for (Orange Bars), <i>SS</i>^<i>70°C</i> (dark red bars) and the relative ratio between and <i>SS</i>^<i>70°C</i> (blue bars). The left y-axis refers to the and <i>SS</i>^<i>70°C</i> and the right y-axis (blue) refers to the relative ratio rc. C) Metabolite; D) Flux.</p

Stochastic internal perturbations.

<p>Histograms accounting for metabolites’ relative steady state variations after stochastically varying V_max. Red: 70°C, Green: 80°C. A) Glc; B) Pyr; C) Gly; D) GA; E) RSD and network elements. # means counts.</p

Metabolome ratios for Glc, D-Gat, KDG, GA and Gly for the WT and knockout.

<p>Measured and simulated metabolite ratios of the ED pathway between PBL 2025ΔSSO3195 (spED = 0) and WT at 80°C.</p

Perturbation of the negative feedforward reaction of the npED branch.

<p>Stochastic perturbations on the feedforward parameters affecting V_GK. A) Pathway diagram of the npED branch with perturbation on the feedforward connection; B) Parametric plot relating Pyr and Gly with feedforward perturbations (red) and without feedforward perturbations (blue); C) Histogram of Pyr steady states with feedforward perturbations (red) and without feedforward perturbations (blue); D) Histogram of Gly steady states with feedforward perturbations (red) and without feedforward perturbations (blue).</p

Robustness to uncertainty and branch deletion.

<p>Histogram accounting for uncertainty and branch deletion at the level of Pyr steady state. Blue bars represent the histogram of Pyr production when the npED branch is set to zero (NP = 0), green bars represent the histogram of Pyr production when the spED branch is set to zero (SP = 0), and red bars represent the histogram of Pyr production for the wild type (WT). A) Relative steady state values. B) Absolute steady state values. # represents counts.</p

Metabolome ratios for Glc, D-Gat, KDG, GA and Gly.

<p>Metabolome ratios calculated with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180331#pone.0180331.e004" target="_blank">Eq 2</a>. Comparison between the metabolomics experiments (metabolites Glc, D-Gat, KDG and Gly) and simulated metabolite concentrations. Relative difference between simulated and experimental levels in blue; simulated calculations in red; experimental values in orange.</p

Stochastic external perturbations.

<p>Histograms of Glc, (A), and Pyr (B), and the relative standard deviations (C) of these three metabolites, at 70°C and 80°C, when changes 50% around its original value. Red bars represent the model at 70°C and green ones at 80°C.</p