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Roles of Ftsn and Dedd in Initiating E. Coli Cell Constriction

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Roles of Ftsn and Dedd in Initiating E. Coli Cell Constriction ROLES OF FTSN AND DEDD IN INITIATING E. COLI CELL CONSTRICTION by BING LIU Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Bing Liu candidate for the degree of PhD*. Committee Chair Arne Rietsch Committee Member Piet de Boer Committee Member Robert Bonomo Committee Member Pieter de Haseth Date of Defense August 21, 2014 *We also certify that written approval has been obtained for any proprietary material contained therein Table of Contents List of Tables ...................................................................................................... iv List of Figures ..................................................................................................... v ABSTRACT ........................................................................................................ vii INTRODUCTION .................................................................................................. 9 Chapter 1. Self-enhanced FtsN activity during the initiation of E. coli cell constriction. ...................................................................................................... 19 INTRODUCTION ......................................................................................................... 19 RESULTS .................................................................................................................... 21 DISCUSSION .............................................................................................................. 29 Chapter 2. Roles for FtsA and the FtsBLQ subcomplex in FtsN-mediated triggering of cell constriction. ......................................................................... 32 INTRODUCTION ......................................................................................................... 32 RESULTS .................................................................................................................... 34 DISCUSSION .............................................................................................................. 53 Chapter 3. DedD assists in initiating cell constriction through an FtsN- independent pathway. ...................................................................................... 59 INTRODUCTION ......................................................................................................... 59 RESULTS .................................................................................................................... 61 DISCUSSION .............................................................................................................. 80 SUMMARY AND FUTURE DIRECTIONS .......................................................... 84 MATERIALS AND METHODS ........................................................................... 88 FIGURES .......................................................................................................... 124 TABLES ............................................................................................................ 181 BIBLIOGRAPHY .............................................................................................. 203 iii List of Tables Table 1 Extragenic suppressors enable growth of cells producing otherwise non- functional FtsN mutant variants. ....................................................................... 181 Table 2 FtsLD93G or FtsBD59H bypasses the requirement for EFtsN. .................. 182 Table 3 Viability of ΔftsN cells harboring (combinations of) suppressing mutations in ftsA, B and/or L. ............................................................................................ 183 Table 4 Requirement for NFtsN in ftsB or ftsL mutant cells lacking EFtsN. ....... 184 Table 5 Suppressing mutations reduce average cell size. ............................... 185 Table 6 FtsBE56A promotes early septal murein synthesis. ............................... 186 Table 7 Localization of GFP-DedD fusions in BL40 cells. ................................ 187 Table 8 Localization of GFP-DedD1-118 mutant variants in BL40 cells. ............. 188 Table 9 Plasmids used in this study. ................................................................. 189 Table 10 E.coli strains used in this study. ......................................................... 199 iv List of Figures FIG. 1 Schematic overview of SR assembly in E. coli. ..................................... 124 FIG. 2 Further definition of the essential domain of FtsN. ................................ 125 FIG. 3 Localization of TTGFP-SFtsN at constriction sites depends on EFtsN and PBP3 activities. ................................................................................................. 127 FIG. 4 Localization of TTGFP-SFtsN and TTGFP in murein amidase mutants. ... 129 FIG. 5 Western analyses of TTGFP-SFtsN. ........................................................ 130 FIG. 6 Model for self-enhanced FtsN activity during the initiation of cell constriction. ....................................................................................................... 132 FIG. 7 Western analyses of periplasmic FtsN fusions. ..................................... 134 FIG. 8 Western analyses of FtsN fusions with internal deletions. ..................... 135 FIG. 9 Genetic screens for extragenic suppressors of nonfunction ftsN alleles. .......................................................................................................................... 136 FIG. 10 Suppressing mutations rescue division of cells producing otherwise non- functional FtsN variants. ................................................................................... 138 FIG. 11 Conserved residues in NFtsN are important for its function and localization. ....................................................................................................... 140 FIG. 12 Western analyses of FtsN1-81 fusions. .................................................. 142 FIG. 13 Viability of ΔftsN cells due to (combinations of) compensating mutations in ftsA, ftsB, and/or ftsL. .................................................................................... 143 FIG. 14 FtsBE56A promotes septal murein synthesis in ftsN+ cells. ................... 145 FIG. 15 Cell shape and lysis phenotypes of various strains in LB’ΔNaCl medium at 42°C, and suppression by ΔftsN. .................................................................. 147 FIG. 16 Lethality of ΔEFtsN-suppressing mutations in ftsB and/or ftsL on LB’ΔNaCl medium at 42°C in the presence of EFtsN. ....................................... 149 FIG. 17 The model for the SR to initiate septal murein synthesis upon initiation of cell constriction. ................................................................................................ 151 FIG. 18 Domain analysis of DedD. ................................................................... 153 FIG. 19 Septal localization of GFP-DedD depends on EFtsN and FtsI. ............ 154 v FIG. 20 Conserved residues in TMDedD are important for DedD’s function in cell division. ............................................................................................................. 156 FIG. 21 Western analyses of GFP-DedD fusions in BL40 cells. ....................... 158 FIG. 22 Western analyses of GFP-DedD1-118 mutants in BL40 cells................. 159 FIG. 23 More EFtsN activity is required for cell division in the absence of DedD. .......................................................................................................................... 161 FIG. 24 Western analyses of DedD and FtsN fusions in ΔdedD ftsNslm117 cells. .......................................................................................................................... 164 FIG. 25 Mutations in TMDedD affect interactions between NDedD and FtsL. .... 165 FIG. 26 BATCH analyses for DedD and FtsN. .................................................. 166 FIG. 27 Combining ΔdedD with ΔponB leads to massive cell lysis. ................. 167 FIG. 28 Model for EFtsN and NDedD in the regulation of sPG synthesis. ......... 169 FIG. 29 Massive cell lysis in ΔdedD ΔponB cells is suppressed by PBP1B or DedD. ................................................................................................................ 170 FIG. 30 Toxicity of excess FtsQ, B, or L molecules in ΔdedD cells. ................. 171 FIG. 31 Suppressing the toxicity of excess FtsQ in ΔdedD cells. ..................... 173 FIG. 32 Overexpression of FtsQ inhibits cell division in the absence of DedD. 174 FIG. 33 Z-ring formation is not affected by overexpression of GFP-FtsQ in the absence of DedD. ............................................................................................. 176 FIG. 34 Overexpression of certain septal ring proteins is toxic to ΔdedD cells. 177 FIG. 35 FtsBE56A suppresses the division defects in ΔdedD cells. .................... 179 FIG. 36 The model for EFtsN and NDedD to initiate septal murein synthesis upon initiation of cell constriction. .............................................................................. 180 vi Roles of FtsN and DedD in Initiating E. Coli Cell Constriction Abstract by BING LIU Upon onset of cell constriction, E. coli cells begin to synthesize new peptidoglycan perpendicularly to the lateral cell wall at
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