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An Analysis Using Fluorescence and Single-Molecule Localization Dynamics of the Min system in Bacillus subtilis An analysis using fluorescence and single- molecule localization microscopy Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie der Ludwig-Maximilians-Universität München Helge Feddersen München, September 2020 Diese Dissertation wurde angefertigt unter der Leitung von Prof. Dr. Marc Bramkamp im Bereich der Mikrobiologie an der Ludwig-Maximilians-Universität München Erstgutachter/in: Prof. Dr. Marc Bramkamp Zweitgutachter/in: Prof. Dr. Kirsten Jung Tag der Abgabe: 18.09.2020 Tag der mündlichen Prüfung: 14.12.2020 ERKLÄRUNG: Ich versichere hiermit an Eides statt, dass meine Dissertation selbständig und ohne unerlaubte Hilfsmittel angefertigt worden ist. Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen Prüfungskommission vorgelegt. Ich habe noch zu keinem früheren Zeitpunkt versucht, eine Dissertation einzureichen oder an einer Doktorprüfung teilzunehmen. Kiel, den 15.01.2021 Helge Feddersen II III „Omnis cellula e cellula.“ - „Jede Zelle [entsteht] aus einer Zelle.“ - Rudolf Virchow, 1855 IV V Contents: I. Abstract .................................................................................................................... X II. Zusammenfassung ............................................................................................... XI III. Abbreviations ................................................................................................. XIII 1. Introduction ............................................................................................................ 1 1.1. Cell division in bacteria ................................................................................ 1 1.1.1. FtsZ and binary fission ..........................................................................................4 1.1.2. Peptidoglycan and the divisome .........................................................................7 1.1.3. Spatio-temporal control of division: Positive regulation ...............................8 1.1.4. Spatio-temporal control of division: Negative regulation .......................... 10 1.1.4.1. Nucleoid occlusion ........................................................................................... 11 1.1.4.2. The Min system in E. coli ............................................................................... 12 In vitro experiments and mathematical modelling of the Min system ......................... 13 1.1.4.3. The Min system in B. subtilis ........................................................................ 13 DivIVA ............................................................................................................................................ 14 MinJ................................................................................................................................................. 16 MinC and MinD........................................................................................................................... 18 1.2. Sporulation of Bacillus subtilis as a second mode of division ............. 19 1.3. Super-resolution microscopy techniques ................................................ 20 1.3.1. Single molecule localization microscopy (SMLM) ....................................... 23 1.3.1.1. Fluorescent proteins and fluorophores in PALM ..................................... 25 1.4. Aims ................................................................................................................ 27 2. Results .................................................................................................................... 28 2.1. Re-characterization of the Min system in B. subtilis ............................. 28 2.1.1. Construction of fluorescent fusions ................................................................ 28 VI Contents 2.1.2. Microscopic analysis reveals dynamics of all Min components ............... 33 2.1.3. Min components affect each other in their dynamics ................................ 36 2.1.4. Mathematical model of the B. subtilis Min system ..................................... 39 2.1.4.1. Quantification of Min proteins ..................................................................... 40 2.1.4.2. A reaction-diffusion model of the Bacillus Min system .......................... 41 2.1.5. SMLM analysis of the Min system .................................................................. 43 2.1.5.1. SMLM reveals apparent clustering of Min proteins ................................ 43 2.1.5.2. Cluster analysis of the Min system ............................................................. 46 2.2. Establishing of SPT ...................................................................................... 49 2.2.1. Sample preparation for SPT imaging .............................................................. 49 2.2.2. Optimization of imaging conditions for SPT ................................................. 51 2.2.3. Choosing the data analysis method for particle tracking .......................... 52 2.2.4. SPT of the B. subtilis Min system ..................................................................... 56 2.2.4.1. Analysis of Min dynamics through MSD analysis................................... 56 2.2.4.2. Analysis of Min dynamics through jump distance analysis .................. 59 2.3. Optimization of mNeonGreen PALM imaging ....................................... 62 2.3.1. Finding the optimal laser intensity for mNeonGreen imaging ................. 63 2.3.2. Characterization of mNeonGreen and comparison to Dronpa ................ 64 2.3.3. Post-processing steps increase mNeonGreen PALM image fidelity ........ 66 2.3.4. PALM sample preparation optimization ........................................................ 69 3. Discussion ............................................................................................................. 71 3.1. The B. subtilis Min system .......................................................................... 71 3.1.1. Construction of functional fluorescent fusions ............................................ 72 3.1.2. B. subtilis Min proteins are dynamic and form clusters ............................. 74 3.1.2.1. FRAP and quantification of the Min proteins ........................................... 74 3.1.2.2. SMLM analysis and a model for the B. subtilis Min proteins ............... 76 3.2. SPT analysis of MinD and DivIVA ............................................................. 81 VII Contents 3.2.1. Technical aspects of SPT in bacterial cells ..................................................... 81 3.2.2. Subpopulations of MinD and DivIVA in SPT ................................................ 84 3.3. Bacterial PALM with mNeonGreen .......................................................... 86 3.4. Conclusion and outlook .............................................................................. 89 4. Materials and Methods ....................................................................................... 91 4.1. Reagents ......................................................................................................... 91 4.2. Oligonucleotides, plasmids and bacterial strains .................................. 91 4.3. Strain construction ....................................................................................... 97 4.3.1. Golden Gate assembly ........................................................................................ 97 4.3.2. Classical cloning ................................................................................................. 102 4.4. Media and growth conditions .................................................................. 103 4.5. Molecular biological methods ................................................................. 104 4.5.1. DNA extraction from E. coli and B. subtilis cells......................................... 104 4.5.2. DNA amplification ............................................................................................. 104 4.5.3. Separation and purification of nucleic acids ............................................... 104 4.5.4. Quantification and sequencing of nucleic acids ......................................... 105 4.5.5. Enzymatic modification of nucleic acids ...................................................... 105 4.5.6. Transformation of E. coli and B. subtilis cells .............................................. 105 4.6. Microscopy .................................................................................................. 106 4.6.1. Fluorescence microscopy .................................................................................. 106 4.6.1.1. FRAP analysis.................................................................................................. 106 4.6.2. Photoactivated localization microscopy (PALM) ....................................... 108 4.6.2.1. Sample preparation ....................................................................................... 108 4.6.2.2. Imaging conditions and individual optimization ................................... 109 4.6.2.3. Data analysis ................................................................................................... 109 4.7. Mathematical modelling of the Min system ......................................... 111 4.7.1. Reaction-diffusion equations .......................................................................... 111 VIII Contents
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