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Systems Biology of the Central Metabolism of Streptococcus Pyogenes

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Systems Biology of the Central Metabolism of Streptococcus Pyogenes Systems biology of the central metabolism of Streptococcus pyogenes Jennifer Levering Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carlo University of Heidelberg, Germany for the degree of Doctor of Natural Science presented by MSc Jennifer Levering born in Wesel Oral-examination: 2011/09/06 Systems biology of the central metabolism of Streptococcus pyogenes Referees: Prof. Dr. Ursula Kummer Prof. Dr. Thomas Höfer So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das Möglichste getan hat. Johann Wolfgang von Goethe Contents List of Figuresv List of Tables viii List of Abbreviations xii Summary xiii 1 Introduction1 1.1 Streptococcus pyogenes ..........................2 1.1.1 Interactions between pathogen and host............3 1.1.2 Metabolic capabilities......................7 1.1.3 Studied kinetics and primary metabolism............9 1.2 Modelling strategies and existing glycolytic models.......... 12 1.3 Project and cooperation partners.................... 14 1.4 Goals of the thesis............................ 17 2 Materials and Methods 19 2.1 Experimental data............................ 20 2.1.1 Bacterial strains.......................... 20 2.1.2 Construction of recombinant vectors and mutant strains... 20 2.1.3 CDM-LAB medium........................ 21 2.1.4 Fermentation experiments.................... 21 2.1.5 Glucose-pulse experiments.................... 22 2.1.6 Kinetic measurements of individual enzymes.......... 23 2.1.7 Substrate utilisation assays.................... 24 2.1.8 Calculation of specific ATP synthesis rates........... 25 2.1.9 Amino acid leave-out experiments................ 25 i 2.2 Dynamic modelling............................ 26 2.2.1 COPASI.............................. 26 2.2.2 Ordinary differential equations................. 27 2.2.3 Kinetics.............................. 28 2.2.4 Simulation............................. 30 2.2.5 Parameter estimation....................... 30 2.2.6 Local sensitivity analysis..................... 31 2.2.7 Global sensitivity analysis.................... 32 2.2.8 Steady state analysis....................... 33 2.2.9 Metabolic control analysis.................... 33 2.3 Genome-scale modelling......................... 35 2.3.1 AUTOGRAPH and INPARANOID............... 35 2.3.2 Manual curation......................... 36 2.3.3 Flux balance analysis....................... 37 3 Results 39 3.1 Experimental results........................... 40 3.1.1 Construction of recombinant vectors and mutant strains... 40 3.1.2 Fermentation experiments.................... 41 3.1.3 Glucose-pulse experiments.................... 46 3.1.4 Kinetic parameters of individual enzymes............ 48 3.1.5 Substrate utilisation assays.................... 49 3.1.6 Calculation of specific ATP synthesis rates........... 49 3.1.7 Amino acid leave-out experiments................ 49 3.2 Kinetic model of S. pyogenes ....................... 55 3.2.1 Setting up the model....................... 55 3.2.2 The role of phosphate...................... 62 3.2.3 Resulting fit............................ 64 3.2.4 Sensitivity analysis........................ 71 3.3 Comparison between S. pyogenes and L. lactis ............. 76 3.3.1 Kinetic model of L. lactis .................... 76 3.3.2 Topological and regulatory differences............. 79 3.3.3 Comparative systems biology of L. lactis and S. pyogenes gly- colysis............................... 82 3.4 Oscillations caused by the stoichiometry................ 85 ii 3.4.1 Finding oscillations in the model................ 85 3.4.2 Results............................... 86 3.5 Reconstructing the metabolic network of S. pyogenes ......... 88 3.5.1 Metabolic pathways incorporated in the model......... 89 3.5.2 Orthology detection........................ 89 3.5.3 Manual curation......................... 90 3.5.4 Filling gaps............................ 92 3.5.5 Characteristics of S. pyogenes .................. 92 3.5.6 Results from flux balance analysis................ 100 3.5.7 Topological comparison with L. lactis and L. plantarum ... 109 4 Discussion 111 4.1 Kinetic model............................... 112 4.1.1 Model set-up, topology and regulation............. 112 4.1.2 The role of phosphate...................... 117 4.1.3 Parameter estimation and resulting fit............. 117 4.1.4 Sensitivity analysis........................ 119 4.2 Comparison to L. lactis ......................... 121 4.2.1 Kinetic model of L. lactis .................... 121 4.2.2 Role of phosphate in L. lactis and S. pyogenes ......... 122 4.3 Genome-scale model........................... 124 4.3.1 Orthology detection........................ 124 4.3.2 Network composition....................... 125 4.3.3 Simulation of experimental data................. 126 4.3.4 Predictions from the model................... 129 4.3.5 Differences and similarities between S. pyogenes and related bacteria.............................. 130 4.4 Discussion of thesis goals......................... 132 5 Outlook 135 Bibliography 137 Acknowledgements 152 iii A Glycolytic model of S. pyogenes 155 A.1 Kinetic parameters and initial concentrations.............. 156 A.2 Rate laws, differential equations and moiety conservation....... 162 A.2.1 Rate laws............................. 162 A.2.2 Differential equations and moiety conservation......... 166 A.3 Results from sensitivity analysis..................... 168 B Genome-scale model of S. pyogenes 179 B.1 Compounds................................ 180 B.2 Reactions................................. 188 B.3 Constraints................................ 198 B.4 Simulation results............................. 203 iv List of Figures 3.1 Overview of molecular interactions of S. pyogenes glycolysis..... 60 3.2 Metabolic profiles and model simulations of glucose-pulse experi- ments in S. pyogenes ........................... 66 3.3 Model simulations and experimental data of glucose-pulse experi- ments in S. pyogenes ........................... 68 3.4 Global sensitivity analysis results for the Vmax of the Pi transporter on FBP.................................. 72 3.5 Maximal global parameter sensitivities on FBP and Pi ........ 73 3.6 Overview of molecular interactions of L. lactis glycolysis....... 78 3.7 Metabolic profiles and model simulations of glucose-pulse experi- ments in L. lactis ............................. 80 3.8 13C- and 31P-NMR time-series profiles and model simulations of glucose- pulse experiments in L. lactis ...................... 81 3.9 Model simulations of intracellular phosphate levels after a 20 mM glucose-pulse in L. lactis ......................... 83 3.10 Model simulations of glycolytic oscillations............... 87 v List of Tables 1.1 Overview of all participating institutions and their roles within the SysMO-LAB project........................... 16 3.1 Maximal specific growth rates of S. pyogenes wild-type and ldh- knock-out................................. 40 3.2 Measured OD and dry weight of S. pyogenes wild-type and ldh-knock- out..................................... 41 3.3 Relative flux distribution in S. pyogenes ................ 42 3.4 Product concentrations in S. pyogenes wild-type strain........ 43 3.5 Product concentrations in S. pyogenes ldh-negative mutant...... 43 3.6 Amino acid concentrations in S. pyogenes wild-type strain...... 44 3.7 Amino acid concentrations in S. pyogenes ldh-negative mutant.... 45 ex 3.8 Metabolite concentration time-series in S. pyogenes for 0 mM Pi .. 47 ex 3.9 Metabolite concentration time-series in S. pyogenes for 10 mM Pi . 47 ex 3.10 Metabolite concentration time-series in S. pyogenes for 50 mM Pi . 48 3.11 Substrate utilisation of S. pyogenes wild-type and its ldh-knock-out mutant on different carbon sources................... 50 3.12 Specific ATP synthesis rates of S. pyogenes .............. 51 3.13 Final OD after 12 h growth of S. pyogenes in full CDM-LAB medium and medium with amino acid leave-outs................. 51 3.14 Essential amino acids for growth of S. pyogenes in CDM-LAB medium based on our experimental data..................... 54 3.15 Rate of medium supply and influx of glucose, acetate and phosphate during continuous cultivation...................... 61 mM 3.16 Velocity constants (in s ) of the steady state model......... 69 3.17 Initial metabolite as well as simulated and measured steady state end-product concentrations (mM) of the steady state model..... 70 vii 3.18 Predicted growth rates (h-1) of S. pyogenes ............... 103 3.19 Simulated growth rates of S. pyogenes wild-type strain grown in full CDM-LAB medium and medium with amino acid leave-outs..... 105 3.20 Essential amino acids for growth of S. pyogenes in CDM-LAB medium compared to literature data....................... 108 mM A.1 Velocity constants (in s )........................ 156 A.2 Reversible processes: Keq ......................... 157 A.3 Michaelis constants: Km (mM)...................... 157 A.4 Allosteric regulation binding constants (mM).............. 160 A.5 Hill coefficients.............................. 161 A.6 Initial concentrations (mM)....................... 161 A.7 Parameter sensitivities on FBP..................... 168 A.8 Parameter sensitivities on Pi ....................... 171 A.9 Parameter sensitivities on the PFK flux................ 173 A.10 Parameter sensitivities on the PTS flux................. 176 B.1 Compounds included in the genome-scale model............ 180 B.2 Reactions included
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