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Chapter 1 on Page 17) UC San Diego UC San Diego Electronic Theses and Dissertations Title Protein localization and peptidoglycan hydrolysis during engulfment in Bacillus subtilis Permalink https://escholarship.org/uc/item/4wn6d926 Author Aung, Stefan Denis Publication Date 2007 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Protein localization and peptidoglycan hydrolysis during engulfment in Bacillus subtilis A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Stefan Denis Aung Committee in charge: Professor Kit Pogliano, Chair Professor Doug Bartlett Professor Michael Burkart Professor Partho Ghosh Professor Lorraine Pillus 2007 Copyright Stefan Denis Aung, 2007 All rights reserved. The Dissertation of Stefan Denis Aung is approved, and it is acceptable in quality and form for publication on microfilm: Chair University of California, San Diego 2007 iii Dedication To my wife, Carissa, and my parents, Dr. Louis and Maureen Aung, who have always supported me in all my endeavors. Thank you for raising me the right way, and for loving me unconditionally. iv Table of Contents Signature Page ……………………………………………………………………… iii Dedication …………………………………………………………………………... iv Table of Contents ……………………………………………………………………. v List of Symbols and Abbreviations ………………………………………………… ix List of Figures...……………………………………………………………………... xi List of Tables .………………………………………………………………………xiv Acknowledgements ………………………………………………………………….xv Curriculum Vitae ...……………………………………………………………….. xvii Abstract ………………………………………………………………………........xviii I. CHAPTER ONE: Introduction……………..………………………………………...1 A. Survival strategies.................................................................................................... 3 B. Engulfment - The beginning stages of sporulation ………………………………. 7 C. Engulfment - What is known?................................................................................ 10 D. A potential role for peptidoglycan hydrolases ....................................................... 15 E. Two modules mediate membrane migration during engulfment ........................... 21 II. CHAPTER TWO: Dual localization pathways for the engulfment proteins during Bacillus subtilis sporulation ………………………………….…………………….. 30 A. Summary ………………………………………………………………………... 31 B. Introduction …………………………………………………………………...… 31 C. Results …………………………………………………………………………... 32 1. Localization of SpoIIB ………………………………………………………... 32 2. Interactions between the DMP proteins …………………………………….… 34 v 3. Localization of SpoIIM and SpoIIP to the septum partially depends on SpoIIB ………………………………………………………………………… 35 4. Rescue of the synergistic engulfment defect of SpoIIP-GFP, spoIIB strains … 35 5. Colocalization of SpoIIB with FtsZ and SpoIIM and SpoIIP ………………… 35 6. Evidence for a SpoIIB-independent targeting pathway ………………………. 35 7. Elimination of both the primary (SpoIIB) and secondary (SpoIVFAB) localization pathways blocks engulfment at the stage of septal thinning …….. 38 D. Discussion ………………………………………………………………………. 38 E. Experimental procedures ………………………………………………………... 40 1. Bacterial strains, genetic manipulations and growth condtions ………………. 40 2. Construction of m-Cherry fusions ……………………………………………. 41 3. Microscopy and image analysis ………………………………………………. 41 4. Antibody production ………………………………………………………….. 41 5. Co-immunoprecipitation and Western blotting ………………………………. 41 6. Immunofluorescence microscopy …………………………………………….. 41 F. Acknowledgements ……………………………………………...……………… 42 G. References ………………………………………………………..………….….. 42 H. Supplemental data …………………………………………………...………….. 44 I. Acknowledgements ................................................................................................ 47 III. CHAPTER THREE: A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore ……………………….………..…… 48 A. Abstract ………………………………………………………………………… 49 B. Introduction …………………………………………………………………….. 49 C. Results ………………………………………………………………………….. 50 1. A genetic screen for membrane migration-defective mutants ……………….. 50 2. Localized mutagenesis of spoIID …………………………………………….. 52 3. Localization of the mother cell-expressed engulfment proteins ……………... 53 vi 4. SpoIID is a peptidoglycan hydrolase ………………………………………… 54 5. The leading edge of the engulfing membrane advances adjacent to the cell wall …………………...………………………………………………….. 55 D. Discussion ……………………………………………………………………… 55 E. Materials and methods ………………………………………………………….. 56 1. Bacterial strains, genetic manipulations, and growth conditions ….………….. 56 2. Localized mutagenesis of spoIID ………………………………….………….. 57 3. Isolation of spoIIP95-2 ……………………...…………………….………….. 57 4. Construction of GFP fusions ……………………………………....…………. 57 5. Microscopy and image analysis ……...…………………………….…………. 58 6. Overexpression and purification of His-SpoIID and His-SpoIIP …………….. 58 7. Renaturing gel electrophoresis for cell wall hydrolytic activity ……………… 58 8. Electron microscopy ………………………………………………………….. 58 F. Acknowledgements ……….………………………………………….………..… 58 G. References ………………………………………………………………………. 59 H. Acknowledgements ............................................................................................... 61 IV. CHAPTER FOUR: Mutational analysis of the endopeptidase SpoIID in Bacillus subtilis …………………………………………………………………………..….. 62 A. Abstract .................................................................................................................. 63 B. Introduction ............................................................................................................ 64 C. Materials and Methods........................................................................................... 70 1. Bacterial strains, genetic manipulations, and growth conditions....................... 70 2. Construction of His6-SpoIID............................................................................. 70 3. Site-directed mutagenesis .................................................................................. 71 4. Microscopy and image analysis ......................................................................... 71 5. Overexpression and purification of His-SpoIID and mutant proteins ............... 72 6. Renaturing gel electrophoresis for cell wall hydrolytic activity (Zymography).73 vii 7. Cell wall binding................................................................................................ 73 8. Spore titer assay ................................................................................................. 74 D. Results.................................................................................................................... 76 1. Site-directed mutagenesis of SpoIID ................................................................. 76 2. Peptidoglycan hydrolysis is necessary for engulfment...................................... 81 3. Sporulation defective mutants............................................................................ 81 4. SpoIID is an endopeptidase ............................................................................... 87 E. Discussion............................................................................................................... 92 F. Acknowledgements................................................................................................. 96 V. CHAPTER FIVE: Concluding Discussion ………………………….…………….. 97 VI. APPENDIX: Crystallization Trials of SpoIID ….…………………..……..………110 A. Introduction.......................................................................................................... 111 B. Results .................................................................................................................. 113 1. Purification and crystallization trials of first generation construct, His6-SpoIID (27-343)........................................................................................ 113 2. Limited Proteolysis ........................................................................................... 119 3. Second generation construct, His6-TEV-SpoIID (56-343) ............................... 123 4. Purification of third generation construct, SpoIID (56-343) ............................ 125 5. Crystallization trials of third generation construct, SpoIID (56-343)............... 127 6. Discussion ......................................................................................................... 130 C. Acknowledgements .............................................................................................. 132 VII. REFERENCES ………………………………………………………………….. 133 viii List of Symbols and Abbreviations C celsius dI deionized DAPI 4’, 6-diamidino-2-phenylindole DMSO dimethylsulfonyloxide DNA deoxyribonucleic acid DNAse I deoxyribonuclease I DSM Difco sporulation media Dpm meso-diaminopimelic acid EtOH ethanol FM 4-64 red fluorescent membrane stain g gram GFP green fluorescent protein (derived from Aequorea Victoria) HPLC high performance liquid chromatography hr./hrs. hour/hours IPTG isopropyl-β-D-thiogalactopyranoside Kan kanamycin KOH potassium hydroxide M molarity mA milliamp mg milligram min minute ml/mls milliliter/milliliters ix mM millimolar NaCl sodium chloride NAG N-acetylglucosamine NAM N-acetylmuramic acid nm nanometer OD optical density O/N overnight PAGE polyacrylamide gel electrophoresis PMSF phenylmethylsulfonyl flouride rpm revolutions per minute RT room temperature
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