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Metabolic and Genomic Annotations in Halophilic Archaea

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Metabolic and Genomic Annotations in Halophilic Archaea Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Metabolic and Genomic Annotations in Halophilic Archaea Tanja Margret Oberwinkler aus Fürstenfeldbruck 2011 Erklärung Diese Dissertation wurde im Sinne von Paragraph 13 Abs. 3 bzw. 4 der Promotion- sordnung vom 29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. Dr. Dieter Oesterhelt betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe angefertigt. München, am 26.7.2011 Tanja Oberwinkler Dissertation eingereicht am: 26.7.2011 1. Gutachter: Prof. Dr. Dieter Oesterhelt 2. Gutachter: Prof. Dr. Helga Stan-Lotter Mündliche Prüfung am: 17. Oktober 2011 Zur lieben Erinnerung an meine Oma Antonia Bernthaler To see a world in a grain of sand And a heaven in a wild flower, Hold infinity in the palm of your hand And eternity in an hour. William Blake, ’Auguries of Innocence’, 1803 vi Contents Summary xix 1 Background 1 1.1 Introduction to halophilic archaea as model organisms . .1 1.1.1 Diversity of halophilic archaea . .1 1.1.2 Ecological distribution of halophilic archaea . .2 1.1.3 Why use halophilic archaea as model organisms? . .4 1.2 Physiology and metabolism . .5 1.2.1 Adaptation to and protective mechanisms in extreme environments5 1.2.1.1 Protections against dehydration . .5 1.2.1.2 Adaptation to oxygen deficiency . .6 1.2.1.3 Protections against photo-oxidation . .7 1.2.2 Metabolic analysis . .7 1.3 Motivation and objectives . .9 2 Material and methods 11 2.1 General materials . 11 2.1.1 Instruments . 11 2.1.2 Chemicals . 11 2.1.3 Kits and enzymes . 11 2.1.4 Software . 11 2.2 Microbiological methods . 11 2.2.1 Strains and culture conditions . 11 2.2.1.1 Overview of strains . 11 2.2.1.2 Culturing and storage of Hbt. salinarum, Nmn. pharaonis and Nmn. moolapensis strains . 14 2.2.2 Media . 14 2.2.2.1 Media for Hbt. salinarum ................. 14 2.2.2.2 Minimal medium for Nmn. pharaonis .......... 16 2.2.2.3 Modified growth medium for Haloarchaea (MGM) . 16 2.2.2.4 Synthetic medium ’DBCM2’ for Nmn. moolapensis .. 16 2.3 Molecular biological methods . 18 2.3.1 Preparation of unpurified (’crude’) DNA from halobacterial cells 18 2.3.2 Gel-electrophoresis of DNA . 18 2.3.3 Isolation of DNA fragments from agarose gels . 19 vii Contents 2.3.4 Determination of DNA concentration . 19 2.3.5 Polymerase chain reaction (PCR) . 19 2.3.6 Sequencing of DNA . 19 2.3.7 Isolation of total RNA . 20 2.3.8 DNase digestion of extracted RNA . 20 2.3.9 DNA microarrays for gene expression profiling . 21 2.3.9.1 Synthesis of fluorescent-labeled cDNA . 21 2.3.9.2 Production and pretreatment of microarray slides . 22 2.3.9.3 Prehybridization and hybridization of microarrays . 22 2.3.9.4 Washing of the slides . 22 2.3.10 Reverse Transcription PCR (RT-PCR) . 23 2.3.10.1 Synthesis of cDNA . 23 2.3.10.2 Gel extraction and DNA sequencing . 24 2.4 Protein chemical methods . 25 2.4.1 Isotope coded protein labeling (ICPL) . 25 2.4.1.1 Isolation of cytosolic proteins of Hbt. salinarum .... 25 2.4.1.2 Reduction, alkylation and nicotinoylation . 25 2.4.2 Membrane isolation by sucrose gradient . 26 2.4.3 Protein precipitation by trichloroacetic acid (TCA) . 26 2.4.4 SDS-Polyacrylamide Gel-Electrophoresis (SDS-PAGE) . 26 2.4.4.1 ICPL samples . 26 2.4.4.2 Analysis of proteins from sucrose gradient bands . 27 2.4.5 Tryptic in-gel-digestion and desalting of protein samples after SDS-PAGE . 27 2.4.5.1 ICPL labeled protein samples . 27 2.4.5.2 Proteins from sucrose gradient . 27 2.5 Analytical methods . 28 2.5.1 Amino acid analysis of Hbt. salinarum ............... 28 2.5.1.1 Calculations of the intracellular change of concentra- tion of ornithine (∆orn) and arginine (∆arg) per cell . 28 2.5.2 Stable Isotope Labeling in Nmn. pharaonis with 13C carbon sources 29 2.5.3 Preparation of lipids . 29 2.5.3.1 Cell harvest and disruption . 29 2.5.3.2 Lipid extraction . 29 2.5.3.3 Extraction of polar lipids by acetone precipitation . 30 2.5.3.4 Cleavage of ether lipids with boron trichloride . 30 2.5.4 Preparation of amino acids . 30 2.5.4.1 Hydrolysis . 31 2.5.4.2 Derivatization . 31 2.5.5 Isolation of intracellular solutes . 31 2.5.6 Thin layer chromatography (TLC) . 32 2.5.6.1 Analytical TLC . 32 viii Contents 2.5.6.2 Preparative TLC with iodine staining . 32 2.5.7 Mass spectrometry . 32 2.5.7.1 Analysis of extracted lipids by negative-ion electro- spray ionization (ESI) mass spectrometry (MS) . 32 2.5.7.2 Analysis of alkyl chains and amino acids with gas chro- matography mass spectrometry (GC/MS) . 33 2.5.7.3 Analysis of proteins extracted from sucrose gradient with NanoLC-MS/MS . 33 2.5.7.4 Analysis of nicotinoylated proteins (ICPL) . 34 2.5.8 Nuclear magnetic resonance . 35 2.5.8.1 NMR measurements of extracted lipids . 35 2.5.8.2 NMR measurements of extracted osmolytes . 36 2.6 Bioinformatics environment . 36 2.6.1 Data analysis of microarrays . 36 2.6.2 Carbon fate modeling in Nmn. pharaonis ............. 36 2.6.3 Bioinformatic analysis . 37 2.6.4 Genome annotation of Nmn. moolapensis ............. 37 2.6.4.1 Genome sequencing and assembly . 37 2.6.4.2 Gene prediction and annotation . 37 3 Lipid metabolism in halophilic Archaea 39 3.1 Introduction . 39 3.1.1 Archaeol - the basic structure of all archaeal membrane lipids . 41 3.1.2 Polar lipids . 42 3.1.3 Biosynthetic pathways of lipids . 42 3.1.3.1 Synthesis of the glycerol phosphate backbone from di- hydroxyacetone phosphate (DHAP) . 42 3.1.3.2 Isoprenoids are synthesized via the mevalonate pathway 44 3.2 Results and discussion . 46 3.2.1 Lipidomics in Hbt. salinarum, Nmn. pharaonis and Nmn. moola- pensis ................................ 46 3.2.1.1 Analytical and preparative TLC of lipid extracts iden- tified different glycolipids . 46 3.2.1.2 Identification of archaeal lipids by negative-ion ESI- mass spectrometry . 48 3.2.2 Stable isotope labeling of isoprenoid chains . 58 3.2.2.1 Assignment of all carbons from the isoprenoid chain by natural abundance 13C NMR . 58 3.2.2.2 13C NMR spectra resulting from differently labeled car- bon sources . 62 3.2.2.3 GC/MS measurements of cleaved isoprenoids supported NMR data . 77 ix Contents 3.3 Conclusions . 83 4 Amino acid metabolism in halophilic Archaea 85 4.1 Introduction . 85 4.1.1 Overview of the haloarchaeal amino acid metabolism . 85 4.1.1.1 Glutamate family (glutamate, glutamine, proline, argi- nine) . 85 4.1.1.2 Aspartate family (aspartate, alanine, asparagine, thre- onine, methionine, lysine) . 86 4.1.1.3 Serine family (serine, glycine, cysteine) . 86 4.1.1.4 Branched-chain amino acids (valine, leucine, isoleucine) 87 4.1.1.5 Aromatic amino acids (phenylalanine, tyrosine, tryp- tophan, histidine) . 87 4.1.2 Carbon fate maps for analysis in metabolic labeling . 87 4.1.3 Arginine fermentation - a unique way to gain energy . 88 4.2 Results and discussion . 89 4.2.1 Distribution of carbons via TCA cycle indicated non-classical amino acid biosynthesis pathways in Nmn. pharaonis ...... 89 4.2.1.1 Incorporation of the label in single amino acids derived from 1-13C- or 2-13C-acetate . 91 4.2.2 Carbon fate modeling in BNet resulted in mixed label types of amino acids . 96 4.2.2.1 Cysteine biosynthesis as an example of a carbon fate map............................ 97 4.2.3 Few alternative carbon sources - other than acetate - support growth of Nmn. pharaonis in minimal medium . 101 4.2.4 Characterization of the arginine/ornithine metabolism in Hbt. sa- linarum ............................... 103 4.2.4.1 Hbt. salinarum can grow without arginine and ornithine in synthetic medium . 103 4.2.4.2 Amino acid uptake experiments with the wild-type Hbt. - salinarum ......................... 105 4.2.4.3 Experiments with a deletion strain verified ArcD as the main arginine uptake system . 106 4.2.4.4 Analysis of arginine fermentation by RNA microarrays and Isotope Coded Protein Labeling (ICPL) . 108 4.3 Conclusions . 111 5 Carbon fixation in halophilic Archaea 113 5.1 Introduction . 113 5.1.1 Calvin cycle (Calvin-Benson-Bassham cycle) . 113 5.1.2 Reductive acetyl-CoA pathway . 114 x Contents 5.1.3 Fatty acid degradation via propionyl CoA - 3-hydroxypropionate cycle . 115 5.1.4 Reductive citrate cycle . 116 5.1.5 CO2 fixation in halophilic Archaea . 117 5.2 Results and discussion . 118 5.2.1 Homology comparison in archaeal genomes . 118 5.2.1.1 Different potential CO2 fixation pathways were found in halophilic archaea . 119 5.2.1.2 Calvin cycle could be excluded . 119 5.2.1.3 Improbable CO2 fixation by the reductive acetyl-CoA pathway . 123 5.2.1.4 3-hydroxypropionate cycle . 124 5.2.1.5 The reductive citrate cycle is possible in Hbt. salinarum 127 5.2.1.6 Reductive condensation of carbon dioxide by the glycine cleavage system . 129 5.2.2 Stable isotope labeling with 13C carbonate in Nmn. pharaonis . 130 5.2.2.1 NMR spectra showing possible CO2 fixation . 130 5.2.2.2 GC/MS derived data does not confirm carbon incor- poration from CO2 in Nmn.
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