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Reconstruction, Modeling & Analysis of Haloarchaeal Metabolic Networks

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Reconstruction, Modeling & Analysis of Haloarchaeal Metabolic Networks Reconstruction, Modeling & Analysis of Haloarchaeal Metabolic Networks Orland Gonzalez M¨unchen, 2009 Reconstruction, Modeling & Analysis of Haloarchaeal Metabolic Networks Orland Gonzalez Dissertation an der Fakult¨at f¨ur Mathematik, Informatik und Statistik der Ludwig-Maximilians-Universit¨at M¨unchen vorgelegt von Orland Gonzalez aus Manila M¨unchen, den 02.03.2009 Erstgutachter: Prof. Dr. Ralf Zimmer Zweitgutachter: Prof. Dr. Dieter Oesterhelt Tag der m¨undlichen Pr¨ufung: 21.01.2009 Contents Summary xiii Zusammenfassung xvi 1 Introduction 1 2 The Halophilic Archaea 9 2.1NaturalEnvironments............................. 9 2.2Taxonomy.................................... 11 2.3PhysiologyandMetabolism.......................... 14 2.3.1 Osmoadaptation............................ 14 2.3.2 NutritionandTransport........................ 16 2.3.3 Motility and Taxis ........................... 18 2.4CompletelySequencedGenomes........................ 19 2.5DynamicsofBlooms.............................. 20 2.6Motivation.................................... 21 3 The Metabolism of Halobacterium salinarum 23 3.1TheModelArchaeon.............................. 24 3.1.1 BacteriorhodopsinandOtherRetinalProteins............ 24 3.1.2 FlexibleBioenergetics......................... 26 3.1.3 Industrial Applications ......................... 27 3.2IntroductiontoMetabolicReconstructions.................. 27 3.2.1 MetabolismandMetabolicPathways................. 27 3.2.2 MetabolicReconstruction....................... 28 3.3Methods..................................... 30 3.3.1 CreatingtheEnzymeInventory.................... 30 vi CONTENTS 3.3.2 ReconstructingtheReactionNetwork................. 34 3.3.3 TheRoleofManualCurationandLiteratureSearch......... 37 3.3.4 Filling the Gaps ............................. 40 3.4NetworkOverview............................... 43 3.5CentralMetabolism............................... 47 3.5.1 Glycolysis/Gluconeogenesis...................... 47 3.5.2 TricarboxylicAcidCycle........................ 50 3.5.3 BypassPathways............................ 50 3.6CatabolicPathways............................... 52 3.6.1 TheRespiratoryChain......................... 52 3.6.2 Glutamate(C5)FamilyofAminoAcids............... 54 3.6.3 Branched-chainAminoAcids..................... 56 3.6.4 Glycine,Serine,ThreonineandAlanine................ 57 3.6.5 AromaticAminoAcids......................... 58 3.6.6 FattyAcids............................... 59 3.7BiosyntheticPathways............................. 60 3.7.1 Nucleotides,AminoAcidsandLipids................. 60 3.7.2 ThePentosePhosphatePathwayandRiboseProduction...... 62 3.7.3 BiosynthesisofAromaticAminoAcids................ 64 4 Systems Analysis of H. salinarum Aerobic Growth and Bioenergetics 69 4.1IntroductiontoMetabolicModels....................... 70 4.1.1 ChemicalKinetics............................ 70 4.1.2 Michaelis-Menten-typeRateLaws................... 71 4.1.3 BiochemicalSystemsTheory...................... 72 4.1.4 LinlogKinetics............................. 73 4.1.5 Constraints-basedModels....................... 73 4.2Methods..................................... 78 4.2.1 MetaboliteConsumptionandProductionEquations......... 78 4.2.2 HybridGenome-scaleFluxBalanceModel.............. 79 4.2.3 CultureConditionsandSamplePreparation............. 80 4.2.4 OxygenConsumption.......................... 81 4.3DeterminationofBiomassComposition.................... 83 4.3.1 AminoAcids.............................. 83 4.3.2 NucleicAcids.............................. 86 CONTENTS vii 4.3.3 SurfaceLayerGlycoproteins...................... 87 4.3.4 MembraneLipids............................ 88 4.4ConsumptionandProductionofNutrients.................. 88 4.5ValidationoftheCriticalPoints........................ 92 4.6CarbonFates.................................. 95 4.6.1 DegradationofEssentialAminoAcids................ 95 4.6.2 InvestigatingBy-productSecretion.................. 97 4.7Bioenergetics.................................. 100 4.8FluxomePrediction............................... 103 4.8.1 Variability Analysis ........................... 103 4.8.2 Nutrient Utilization ........................... 105 5 Phototrophic Growth of H. salinarum 111 5.1GeneralComparisonwithAerobicCase.................... 111 5.2MetaboliteConsumptionandProduction................... 112 5.3EnvironmentalAdaptations.......................... 120 6AnalysisofN. pharaonis Growth 123 6.1ThePolyextremophile............................. 123 6.2MetabolicReconstruction........................... 124 6.2.1 CentralMetabolism........................... 124 6.2.2 NutritionalSelf-sufficiencyandBioenergetics............. 125 6.3Methods..................................... 127 6.3.1 CultureConditionsandSamplePreparation............. 127 6.3.2 AcetateAssay.............................. 128 6.4Biomass..................................... 129 6.5AerobicGrowth................................. 129 6.5.1 GrowthasaFunctionofAcetateandOxygenConsumption.... 129 6.5.2 AnActualCulture........................... 133 6.5.3 SummaryoftheNaphaModel..................... 135 7 Conclusions and Outlook 137 7.1ContributionsofthisThesis.......................... 137 7.2FutureDirections................................ 139 A Reconstructed Networks 143 viii Inhaltsverzeichnis Acknowledgements 198 List of Figures 1.1Petrinetrepresentationoftheoverallstructureofthisthesis........ 2 2.1Examplesofhypersalineenvironments.................... 10 2.2LongtermchangesintheDeadSea...................... 12 2.3 Distribution of halophilic microorganisms ................... 13 2.4 Darkfield micrograph of Haloquadratum walsbyi ............... 14 2.5 Motility structures ............................... 18 3.1 Massive Growth of halophilic archaea in a saltern .............. 25 3.2The3-Dstructureofbacteriorhodopsin.................... 26 3.3MetabolicmapshowingasegmentoftheTCAcycle............. 29 3.4Summaryofstepsleadingtoadraftnetwork................. 31 3.5 A representation of the genome of Halobacterium salinarum ........ 32 3.62DGenomeAnnotation............................ 37 3.7Modifiedmevalonatepathway......................... 39 3.8 Filling the gaps - Coenzyme B12 biosynthesis ................ 41 3.9 The cobalamin cluster of Halobacterium salinarum ............. 42 3.10Thedistributionofreactionsbycategory................... 44 3.11Networkevidence................................ 45 3.12Networkevidencebycategory......................... 47 3.13EMpathway,TCAcycleandsomeassociatedreactions........... 49 3.14ProposedOxidativePhosphorylationPathway................ 53 3.15Catabolismoftheglutamate(C5)familyofaminoacids.......... 55 3.16Catabolismofglycine,serine,threonineandalanine............. 57 3.17Essentialityofarginine............................. 66 3.18ThePentosePhosphatePathwayandriboseproduction........... 67 3.19Biosynthesisofaromaticaminoacids..................... 68 x LIST OF FIGURES 4.1Principlesofconstraint-basedmodeling.................... 74 4.2 A small illustrative network .......................... 75 4.3Dissolutionkineticsofoxygen......................... 82 4.4 Amino acid composition of the Halobacterium salinarum biomass..... 85 4.5Schematicrepresentationofthecellsurfaceglycoprotein.......... 87 4.6 Nutrient consumption and production data from aerobically grown cells . 90 4.7 Parameter (ti,b)explorationforaerobicallygrowncells............ 93 4.8Summaryofnutrientuptakeandincorporationrates(aerobic)....... 96 4.9Carbonconsumptionandbiomassincorporation(aerobic).......... 97 4.10Aminoacidcompositionofbiomassduringaerobicgrowth......... 98 4.11Theoretical(energy-optimal)andactualoxygenconsumptionrates..... 100 4.12SystemenergyinATPequivalent....................... 101 4.13Predictedfluxomeduringlogphase...................... 104 4.14Arginine,prolineandornithinemetabolism.................. 108 5.1Representativegrowthcurves......................... 112 5.2Aminoacidcompositionofbiomassduringphototrophicgrowth...... 113 5.3Comparisonofbiomassaminoacidcomposition............... 114 5.4Nutrientconsumptionandproduction(anaerobic,light)........... 116 5.5Summaryofnutrientuptakeandincorporationrates(phototrophic).... 118 5.6 Total carbon consumption and biomass incorporation (phototrophic) . 119 6.1 Electron microscopic image of Natronomonas pharaonis ........... 123 6.2Thedistributionofreactionsbycategory................... 125 6.3 EM pathway and TCA cycle in Natronomonas pharaonis .......... 126 6.4 Amino acid composition of the Natronomonas pharaonis biomass..... 130 6.5 Biomass amino acid composition comparison with Halobacterium salinarum 131 6.6Maximumgrowthasafunctionofacetateandoxygenconsumption.... 132 6.7Maximumgrowthasafunctionofacetate.................. 132 6.8 Natronomonas pharaonis grownaerobicallyonacetate........... 133 6.9Aerobicgrowthsummary............................ 134 List of Tables 2.1 Genera and species under the order Halobacteriales ............. 15 3.1Top-levelECNumbers............................. 35 3.2Comparisonofgenomeandreconstructionwithotherorganisms...... 44 3.3ReactionsandenzymesinthenetworkbyKEGGdefinedpathways.... 46 3.4Summaryofaminoacidmetabolism...................... 61 4.1CompositionofChemically-definedMedium................. 81 4.2Averagecellularbiomasscomposition..................... 84 4.3Comparisonoftransportequationforms................... 89 6.1 Composition of defined media used for Natronomonas pharaonis ...... 128 A.1 Halobacterium salinarum reactionlist..................... 143 A.2 Natronomonas pharaonis reactionlist....................
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