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Reconstruction, modeling & analysis of haloarchaeal metabolic networks [Elektronische Ressource] / vorgelegt von Orland Gonzalez

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222 pages
Reconstruction, Modeling & Analysis ofHaloarchaeal Metabolic NetworksOrland GonzalezM¨ unchen, 2009Reconstruction, Modeling & Analysis ofHaloarchaeal Metabolic NetworksOrland GonzalezDissertationan der Fakult¨at fur¨ Mathematik, Informatik und Statistikder Ludwig-Maximilians-Universit¨atM¨ unchenvorgelegt vonOrland Gonzalezaus ManilaM¨ unchen, den 02.03.2009Erstgutachter: Prof. Dr. Ralf ZimmerZweitgutachter: Prof. Dr. Dieter OesterheltTag der mundlic¨ hen Prufung:¨ 21.01.2009ContentsSummary xiiiZusammenfassung xvi1 Introduction 12 The Halophilic Archaea 92.1 NaturalEnvironments............................. 92.2 Taxonomy................ 112.3 PhysiologyandMetabolism...... 142.3.1 Osmoadaptation ............................ 142.3.2 NutritionandTransport.... 162.3.3 Motility and Taxis ....... 182.4 CompletelySequencedGenomes........................ 192.5 DynamicsofBlooms.......... 202.6 Motivation................ 213 The Metabolism of Halobacterium salinarum 233.1 TheModelArchaeon.............................. 243.1.1 BacteriorhodopsinandOtherRetinalProteins... 243.1.2 FlexibleBioenergetics ..... 263.1.3 Industrial Applications.................... 273.2 IntroductiontoMetabolicReconstructions......... 273.2.1 MetabolismandMetabolicPathways........ 273.2.2 MetabolicReconstruction ....................... 283.3 Methods................. 303.3.1 CreatingtheEnzymeInventory 30vi CONTENTS3.3.2 ReconstructingtheReactionNetwork.................
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Reconstruction, Modeling & Analysis of
Haloarchaeal Metabolic Networks
Orland Gonzalez
M¨ unchen, 2009Reconstruction, Modeling & Analysis of
Haloarchaeal Metabolic Networks
Orland Gonzalez
Dissertation
an der Fakult¨at fur¨ Mathematik, Informatik und Statistik
der Ludwig-Maximilians-Universit¨at
M¨ unchen
vorgelegt von
Orland Gonzalez
aus Manila
M¨ unchen, den 02.03.2009Erstgutachter: Prof. Dr. Ralf Zimmer
Zweitgutachter: Prof. Dr. Dieter Oesterhelt
Tag der mundlic¨ hen Prufung:¨ 21.01.2009Contents
Summary xiii
Zusammenfassung xvi
1 Introduction 1
2 The Halophilic Archaea 9
2.1 NaturalEnvironments............................. 9
2.2 Taxonomy................ 11
2.3 PhysiologyandMetabolism...... 14
2.3.1 Osmoadaptation ............................ 14
2.3.2 NutritionandTransport.... 16
2.3.3 Motility and Taxis ....... 18
2.4 CompletelySequencedGenomes........................ 19
2.5 DynamicsofBlooms.......... 20
2.6 Motivation................ 21
3 The Metabolism of Halobacterium salinarum 23
3.1 TheModelArchaeon.............................. 24
3.1.1 BacteriorhodopsinandOtherRetinalProteins... 24
3.1.2 FlexibleBioenergetics ..... 26
3.1.3 Industrial Applications.................... 27
3.2 IntroductiontoMetabolicReconstructions......... 27
3.2.1 MetabolismandMetabolicPathways........ 27
3.2.2 MetabolicReconstruction ....................... 28
3.3 Methods................. 30
3.3.1 CreatingtheEnzymeInventory 30vi CONTENTS
3.3.2 ReconstructingtheReactionNetwork................. 34
3.3.3 TheRoleofManualCurationandLiteratureSearch 37
3.3.4 Filling the Gaps.................... 40
3.4 NetworkOverview .................... 43
3.5 CentralMetabolism 47
3.5.1 Glycolysis/Gluconeogenesis ............. 47
3.5.2 TricarboxylicAcidCycle............. 50
3.5.3 BypassPathways........ 50
3.6 CatabolicPathways...................... 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 FatyAcids...................... 59
3.7 BiosyntheticPathways.................. 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
4.1 IntroductiontoMetabolicModels....................... 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
4.2 Methods..................................... 78
4.2.1 MetaboliteConsumptionandProductionEquations 78
4.2.2 HybridGenome-scaleFluxBalanceModel..... 79
4.2.3 CultureConditionsandSamplePreparation............. 80
4.2.4 OxygenConsumption................. 81
4.3 DeterminationofBiomasComposition 83
4.3.1 AminoAcids ................... 83
4.3.2 NucleicAcids........... 86CONTENTS vii
4.3.3 SurfaceLayerGlycoproteins...................... 87
4.3.4 MembraneLipids........ 8
4.4 ConsumptionandProductionofNutrients......... 8
4.5 ValidationoftheCriticalPoints........................ 92
4.6 CarbonFates.............. 95
4.6.1 DegradationofEsentialAminoAcids ....... 95
4.6.2 InvestigatingBy-productSecretion.................. 97
4.7 Bioenergetics.........................100
4.8 FluxomePrediction103
4.8.1 Variability Analysis................103
4.8.2 Nutrient Utilization...........105
5 Phototrophic Growth of H. salinarum 111
5.1 GeneralComparisonwithAerobicCase.........11
5.2 MetaboliteConsumptionandProduction..........12
5.3 EnvironmentalAdaptations.................120
6AnalysisofN. pharaonis Growth 123
6.1 ThePolyextremophile ..................123
6.2 MetabolicReconstruction ..................124
6.2.1 CentralMetabolism124
6.2.2 NutritionalSelf-sufficiencyandBioenergetics.............125
6.3 Methods............................127
6.3.1 CultureConditionsandSamplePreparation....127
6.3.2 AcetateAsay...................128
6.4 Biomass............................129
6.5 AerobicGrowth..129
6.5.1 GrowthasaFunctionofAcetateandOxygenConsumption ....129
6.5.2 AnActualCulture...........................13
6.5.3 SummaryoftheNaphaModel.135
7 Conclusions and Outlook 137
7.1 ContributionsofthisThesis..........................137
7.2 FutureDirections............139
A Reconstructed Networks 143viii Inhaltsverzeichnis
Acknowledgements 198List of Figures
1.1 Petrinetrepresentationoftheoveralstructureofthisthesis........ 2
2.1 Examplesofhypersalineenvironments .................... 10
2.2 LongtermchangesintheDeadSea.. 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
3.2 The3-Dstructureofbacteriorhodopsin 26
3.3 MetabolicmapshowingasegmentoftheTCAcycle.... 29
3.4 Summaryofstepsleadingtoadraftnetwork................. 31
3.5 A representation of the genome of Halobacterium salinarum ........ 32
3.6 2DGenomeAnnotation ................... 37
3.7 Modifiedmevalonatepathway.............. 39
3.8 Filling the gaps - Coenzyme B12 biosynthesis ....... 41
3.9 The cobalamin cluster of Halobacterium salinarum .... 42
3.10Thedistributionofreactionsbycategory................... 44
3.1Networkevidence....................... 45
3.12Networkevidencebycategory..... 47
3.13EMpathway,TCAcycleandsomeasociatedreactions........... 49
3.14ProposedOxidativePhosphorylationPathway....... 53
3.15Catabolismoftheglutamate(C5)familyofaminoacids . 5
3.16Catabolismofglycine,serine,threonineandalanine............. 57
3.17Esentialityofarginine.................... 6
3.18ThePentosePhosphatePathwayandriboseproduction.. 67
3.19Biosynthesisofaromaticaminoacids.......... 68x LIST OF FIGURES
4.1 Principlesofconstraint-basedmodeling.................... 74
4.2 A small illustrative network ...... 75
4.3 Disolutionkineticsofoxygen..... 82
4.4 Amino acid composition of the Halobacterium salinarumbiomas ..... 85
4.5 Schematicrepresentationofthecelsurfaceglycoprotein .......... 87
4.6 Nutrient consumption and production data from aerobically grown cells . . 90
4.7 Parameter (t )explorationforaerobicalygrowncels... 93i,b
4.8 Summaryofnutrientuptakeandincorporationrates(aerobic)....... 96
4.9 Carbonconsumptionandbiomasincorporation(aerobic).......... 97
4.10Aminoacidcompositionofbiomasduringaerobicgrowth 98
4.1Theoretical(energy-optimal)andactualoxygenconsumptionrates.....10
4.12SystemenergyinATPequivalent.......................101
4.13Predictedfluxomeduringlogphase..104
4.14Arginine,prolineandornithinemetabolism.........108
5.1 Representativegrowthcurves .........................12
5.2 Aminoacidcompositionofbiomassduringphototrophicgrowth......113
5.3 Comparisonofbiomasaminoacidcomposition......14
5.4 Nutrientconsumptionandproduction(anaerobic,light)...........16
5.5 Summaryofnutrientuptakeandincorporationrates(phototrophic)....18
5.6 Total carbon consumption and biomass incorporation (phototrophic) . . . 119
6.1 Electron microscopic image of Natronomonas pharaonis ...........123
6.2 Thedistributionofreactionsbycategory..........125
6.3 EM pathway and TCA cycle in Natronomonas pharaonis .126
6.4 Amino acid composition of the Natr pharaonisbiomas .....130
6.5 Biomass amino acid composition comparison with Halobacterium salinarum 131
6.6 Maximumgrowthasafunctionofacetateandoxygenconsumption....132
6.7 Maximumgrowthasafunctionofacetate ..................132
6.8 Natronomonas pharaonisgrownaerobicalyonacetate ..13
6.9 Aerobicgrowthsummary...................134

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