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Multi-Omics Based Characterization of Various Stress Responses in Bacillus Subtilis

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Multi-Omics Based Characterization of Various Stress Responses in Bacillus Subtilis Multi-omics based characterization of various stress responses in Bacillus subtilis Inauguraldissertation zur Erlangung des akademischen Grades Doktor rerum naturalium (Dr. rer. nat.) an der Mathematisch- Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald Vorgelegt von Praveen Kumar Sappa geboren am 05.08.1986 in Tadepalligudem – India Greifswald, 2013 Dekan: Prof. Dr. rer. nat. Klaus Fesser 1. Gutachter: Prof. Dr. rer. nat. Uwe Völker 2. Gutachter: Prof. Dr. Oscar Kuipers Tag der Promotion: 24.04.2014 Praveen Kumar Sappa Table of contents 1. TABLE OF CONTENTS 1. Table of Contents .................................................................................................................................. 3 2. Summary ............................................................................................................................................... 7 3. Introduction ......................................................................................................................................... 11 3.1 Stress responses ........................................................................................................................... 11 3.2 Central carbon metabolism .......................................................................................................... 13 3.2.1 Regulation of CCM ............................................................................................................. 18 3.3 Osmoprotection ........................................................................................................................... 20 3.4 Genomics and Proteomics ........................................................................................................... 25 3.5 Aim of the study .......................................................................................................................... 30 4. Materials and methods ......................................................................................................................... 33 4.1 List of abbreviations .................................................................................................................... 33 4.2 Strains used in the study .............................................................................................................. 34 4.3 Biphasic batch fermentation (performed by Dr. Beate Knoke, Stuttgart) ................................... 34 4.4 Growth conditions for transcriptome analysis by tiling arrays .................................................... 35 4.4.1 Growth at 37°C .................................................................................................................... 35 4.4.2 Growth in the presence of 1.2 M NaCl ................................................................................ 36 4.4.3 Growth at 16°C .................................................................................................................... 37 4.4.4 Growth at 51°C .................................................................................................................... 37 4.4.5 Growth in M9 ...................................................................................................................... 37 4.5 Experiments for optimizing trypsin digestion of protein samples from sporulating B. subtilis .. 38 4.5.1 Growth in DSM media ........................................................................................................ 39 4.5.2 Calculation of percentage of heat resistant endospores (heat kill assay) ............................. 39 4.6 Chemostat based cultivations (performed by Michael Kohlstedt, Braunschweig) ...................... 39 4.7 Sample harvesting for transcriptome analysis ............................................................................. 40 4.8 RNA preparation ......................................................................................................................... 40 4.9 Gene expression profiling ........................................................................................................... 41 4.9.1 Relative gene expression analysis ....................................................................................... 41 4.9.2 Tiling array design ............................................................................................................... 43 4.9.3 Statistical and functional data analysis in Genedata Analyst .............................................. 44 4.10 Protein isolation and estimation .................................................................................................. 44 4.10.1 Sample harvesting and preparation of protein lysate by cell disruption .............................. 44 4.10.2 Determination of protein concentrations by Ninhydrin assay ............................................. 44 4.10.3 Determination of protein concentrations by Bradford assay ............................................... 45 4.11 Proteolysis and purification of proteome samples ....................................................................... 45 4.11.1 Proteolysis by trypsin .......................................................................................................... 46 4.11.2 Purification and in-gel digestion of the proteins ................................................................. 46 3 Praveen Kumar Sappa Table of contents 4.11.3 Desalting of peptides by ZipTip® ....................................................................................... 47 4.12 Mass spectrometry ....................................................................................................................... 47 4.12.1 MS parameters –LTQ Velos Orbitrap ................................................................................. 47 4.12.2 MS parameters –TSQ Vantage ............................................................................................ 47 4.12.3 Data analysis ........................................................................................................................ 48 4.13 Absolute protein quantification by Multiple Reaction Monitoring (MRM) ................................ 49 4.13.1 Design of the QconCATs .................................................................................................... 49 4.13.2 MRM method development ................................................................................................. 49 4.14 Enzyme activity assays (performed by Michael Kohlstedt, Braunschweig) ............................... 57 4.15 Metabolome analysis (performed by Hanna Meyer, Greifswald) ............................................... 57 4.16 13C Metabolic flux analysis (performed by Michael Kohlstedt, Braunschweig) ....................... 59 4.16.1 Visualization of multi-omics data sets and pathway mapping. ........................................... 59 5. Results ................................................................................................................................................. 61 5.1 Trypsin digestion optimization for the samples obtained from sporulating B. subtilis ............... 61 5.2 Multi-omics profiling of B. subtilis during the transition from growing to non-growing state ... 65 5.2.1 Transcriptome profiling of B. subtilis in tri-phase fed-batch fermentation ......................... 65 5.2.2 Multi-omics analysis of B. subtilis in bi-phase batch fermentation..................................... 68 5.3 Whole-transcriptome analysis of B. subtilis at high temperature, low temperature, high salt and during glucose starvation............................................................................................................................. 77 5.4 Multi-omics study of osmostressed- and osmoprotected B. subtilis under glucose limiting conditions .................................................................................................................................................... 81 5.4.1 Analysis of transcriptional changes ..................................................................................... 83 5.4.2 Multi-omics investigation of the central carbon metabolism .............................................. 86 5.4.3 Effect of glycine betaine during exponential growth and osmoprotection .......................... 94 5.4.4 Comparative analysis of the central carbon metabolism during osmostress and osmoprotection at high and low glucose ........................................................................................... 104 5.4.5 Effect of ProH and ProJ over-expression and glutamate supplementation onto the osmo- adaptation of B. subtilis ..................................................................................................................... 108 5.4.6 The effect of glutamate supplementation on osmoadaptation of B. subtilis ...................... 112 6. Discussion ......................................................................................................................................... 114 6.1 Multi-omics profiling of B. subtilis during the transition from growing to non-growing state . 114 6.2 Multi-omics analysis of B. subtilis cultivated in bi-phase batch fermentation .......................... 117 6.3 Whole-transcriptome analysis of B. subtilis at high temperature, low temperature, high salt and during glucose starvation..........................................................................................................................
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