Universität Stuttgart
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Item Open Access Metabolic analysis of carotenoid dynamics and global metabolism in carotenoid mutants of Rhodospirillum rubrum using HPLC/MS methodology(2013) Bóna-Lovász, Judit; Ghosh, Robin (Prof. Dr.)In this study, Rhodospirillum rubrum, a photosynthetic, facultative anaerobic bacterium was investigated as a potentially good candidate for industrial carotenoid production. Due to its high amounts of intracellular membrane (ICM), it provides more possibilities for the storage of carotenoids than E. coli, where only the cytoplasmic membrane is available for sequestration. Since R. rubrum is an anoxygenic phototrophic bacterium, all photosynthetic operators are consequently regulated by oxygen in contrast to aerobic phototrophic microorganisms. The photosynthetic membrane (PM) and carotenoid biosynthesis can only be induced at low (pO2<0.5%) oxygen concentrations. Despite the detailed characterization of the PM there is a paucity of information regarding the biosynthesis and the assembly of the photosynthetic system. Therefore, the wild-type, ST4 (crtD-) and SLYC18 (crtC-crtD-) carotenoid mutants were studied by rapid sampled anaerobic and microaerophilic, dark fed-batch fermentations using fructose-succinate medium. The roles of the regulatory signals - O2 and ubiquinone (UQ) pool - mediating the metabolic pattern and the induction of PM biosynthesis were also investigated in this study. The metabolic pattern showed that strong oxygen and CO2 limitation resulted in extremely slow growth. High-level organic acid excretion and polyhydroxybutyrate (PHB) biosynthesis were observed, reflecting the fully reduced state of the cell. The metabolic profile, induced by the high intracellular acetate concentrations occurring in the initial phase of the growth curve, indicated that the citramalate and the ethylmalonyl pathways were active under anaerobic conditions. Succinate production, presumably via the methylmalonyl pathway, was also observed in absence of oxygen. SLYC18 was found to grow twice as fast as the wild-type under CO2- and oxygen-limited conditions. Under anaerobic conditions SLYC18 contained a two fold higher level of rhodoquinone-10 (RQ) and PHB than the wild-type. Since the high RQ level and the enhanced PHB production show a strong correlation, it is proposed here that the RQ-mediated production of NADPH occurs via a putative NADPH dehydrogenase. In order to investigate the biosynthesis of the PM, the major lipid fractions (fatty acids, phospholipids), as well as carotenoids, isoprenoid quinones and bacteriochlorophyll a (BChla) were measured simultaneously during the fermentation process. Therefore, a single-step extraction with a ternary hexane/methanol/water mixture followed by HPLC with mass spectrometric detection was developed for determination of carotenoids and other non-polar compounds present. The method is suitable for extracting large numbers of samples, which is common in systems biology studies. The procedure was able to determine 18 carotenoids, 4 isoprenoid-quinones, BChla and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The high-resolution time courses of the carotenoid biosynthesis allowed the conversion rate of each carotenogenic reaction to be studied. A strong bottleneck was observed at the reactions of CrtD. Furthermore, the second reactions of all enzymes are significantly slower than the first one. Analysis of the carotenoid dynamics indicated that several parallel carotenoid pathways can be linked at common connection points (hydroxylated carotenoids). The carotenoid biosynthesis was more strongly affected by the low oxygen concentration than the central metabolic pattern. The carotenoid mutants were the most sensitive to oxygen, resulting in growth inhibition and a 33% decrease of anaerobic carotenoid concentration in SLYC18. The carotenogenic reaction sequence was blocked in the wild-type in the presence of oxygen, which resulted in the accumulation of anhydrorhodovibrin instead of the normal major carotenoid, spirilloxanthin. The presence of oxygen clearly affected the activity of CrtD resulting in the opening of two new side-pathways: the alternative spheroidene pathway (4%) in the wild-type and ST4 and the 3,4-didehydrolycopene pathway (6%) in ST4. The existence of the 3,4-didehydrolycopene pathway, which has never been observed in R. rubrum earlier, indicated the ability of CrtI in catalyzing five desaturation steps in the presence of oxygen in the ST4 mutant. The PM biosynthesis showed a strong dependence on the changes of the [UQ]/[UQH2] ratio. Under anaerobic conditions, the metabolism is sensitive to pulses of oxygen, causing temporary inactivation the oxidative tricarboxylic acid cycle and the electron transport chain and changes in the UQ pool redox state. This oscillation pattern was analyzed by system biological methods. The systems biology analysis suggested that the isoprenoids are regulated by an exogenous signal. Considering the metabolic observations in the experiments, the regulatory signal might possibly be the [UQH2]/[UQ] ratio.Item Open Access Rapid sampling of Escherichia coli after changing oxygen conditions reveals transcriptional dynamics(2017) Wulffen, Joachim von; Ulmer, Andreas; Jäger, Günter; Sawodny, Oliver; Feuer, RonnyEscherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression.Item Open Access Basic regulatory principles of Escherichia coli's electron transport chain for varying oxygen conditions(2014) Henkel, Sebastian; Beek, Alexander ter; Steinsiek, Sonja; Stagge, Stefan; Bettenbrock, Katja; Teixeira de Mattos, M. Joost; Sauter, Thomas; Sawodny, Oliver; Ederer, MichaelFor adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.Item Open Access Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol(2012) Feuer, Ronny; Gottlieb, Katrin; Viertel, Gero; Klotz, Johannes; Schober, Steffen; Bossert, Martin; Sawodny, Oliver; Sprenger, Georg; Ederer, MichaelBacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.Item Open Access A rapid method for the extraction and analysis of carotenoids and other hydrophobic substances suitable for systems biology studies with photosynthetic bacteria(2013) Bóna-Lovász, Judit; Bóna, Aron; Ederer, Michael; Sawodny, Oliver; Ghosh, RobinA simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%-100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.Item Open Access Mathematical modeling and simulation of thyroid homeostasis : implications for the Allan-Herndon-Dudley syndrome(2022) Wolff, Tobias M.; Veil, Carina; Dietrich, Johannes W.; Müller, Matthias A.Introduction: A mathematical model of the pituitary-thyroid feedback loop is extended to deepen the understanding of the Allan-Herndon-Dudley syndrome (AHDS). The AHDS is characterized by unusual thyroid hormone concentrations and a mutation in the SLC16A2 gene encoding for the monocarboxylate transporter 8 (MCT8). This mutation leads to a loss of thyroid hormone transport activity. One hypothesis to explain the unusual hormone concentrations of AHDS patients is that due to the loss of thyroid hormone transport activity, thyroxine (T4) is partially retained in thyroid cells. This hypothesis is investigated by extending a mathematical model of the pituitary-thyroid feedback loop to include a model of the net effects of membrane transporters such that the thyroid hormone transport activity can be considered. A nonlinear modeling approach based on the Michaelis-Menten kinetics and its linear approximation are employed to consider the membrane transporters. The unknown parameters are estimated through a constrained parameter optimization. In dynamic simulations, damaged membrane transporters result in a retention of T4 in thyroid cells and ultimately in the unusual hormone concentrations of AHDS patients. The Michaelis-Menten modeling approach and its linear approximation lead to similar results. The results support the hypothesis that a partial retention of T4 in thyroid cells represents one mechanism responsible for the unusual hormone concentrations of AHDS patients. Moreover, our results suggest that the retention of T4 in thyroid cells could be the main reason for the unusual hormone concentrations of AHDS patients.Item Open Access Model-based characterization of inflammatory gene expression patterns of activated macrophages(2016) Rex, Julia; Albrecht, Ute; Ehlting, Christian; Thomas, Maria; Zanger, Ulrich M.; Sawodny, Oliver; Häussinger, Dieter; Ederer, Michael; Feuer, Ronny; Bode, Johannes G.Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.Item Open Access A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen(2014) Ederer, Michael; Steinsiek, Sonja; Stagge, Stefan; Rolfe, Matthew D.; Beek, Alexander tek; Knies, David; Teixeira de Mattos, M. Joost; Sauter, Thomas; Green, Jeffrey; Poole, Robert K.; Bettenbrock, Katja; Sawodny, OliverThe efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.Item Open Access Transition of an anaerobic Escherichia coli culture to aerobiosis: balancing mRNA and protein levels in a demand-directed dynamic flux balance analysis(2016) Wulffen, Joachim von; Sawodny, Oliver; Feuer, RonnyThe facultative anaerobic bacterium Escherichia coli is frequently forced to adapt to changing environmental conditions. One important determinant for metabolism is the availability of oxygen allowing a more efficient metabolism. Especially in large scale bioreactors, the distribution of oxygen is inhomogeneous and individual cells encounter frequent changes. This might contribute to observed yield losses during process upscaling. Short-term gene expression data exist of an anaerobic E. coli batch culture shifting to aerobic conditions. The data reveal temporary upregulation of genes that are less efficient in terms of energy conservation than the genes predicted by conventional flux balance analyses. In this study, we provide evidence for a positive correlation between metabolic fluxes and gene expression. We then hypothesize that the more efficient enzymes are limited by their low expression, restricting flux through their reactions. We define a demand that triggers expression of the demanded enzymes that we explicitly include in our model. With these features we propose a method, demand-directed dynamic flux balance analysis, dddFBA, bringing together elements of several previously published methods. The introduction of additional flux constraints proportional to gene expression provoke a temporary demand for less efficient enzymes, which is in agreement with the transient upregulation of these genes observed in the data. In the proposed approach, the applied objective function of growth rate maximization together with the introduced constraints triggers expression of metabolically less efficient genes. This finding is one possible explanation for the yield losses observed in large scale bacterial cultivations where steady oxygen supply cannot be warranted.Item Open Access IL-1β and TNFα differentially influence NF-κB activity and FasL-induced apoptosis in primary murine hepatocytes during LPS-induced inflammation(2019) Rex, Julia; Lutz, Anna; Faletti, Laura E.; Albrecht, Ute; Thomas, Maria; Bode, Johannes G.; Borner, Christoph; Sawodny, Oliver; Merfort, IrmgardMacrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1β, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1β induces increased caspase-3 activity but keeps cells alive. We now report that IL-1β and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1β and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1β and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection.