Force generation in dividing E. coli cells: A handles-on approach using optical tweezers
Verhoeven, G.S.
Citation
Verhoeven, G. S. (2008, December 2). Force generation in dividing E. coli cells: A handles- on approach using optical tweezers. Retrieved from https://hdl.handle.net/1887/13301
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13301
Contents
Chapter 1: Introduction...11
Measuring and exerting forces with optical tweezers... 14
Force probe attachment to the site of division ... 16
Outline of this thesis... 18
Chapter 2: Force-induced cell shaping of bacteria? ...19
The PG cell wall ... 21
Turgor pressure... 22
The cytoskeleton: FtsZ ... 23
The cytoskeleton: MreB ... 25
PBPs and PG hydrolases: making and breaking bonds in the sacculus ... 25
Cell division ... 26
Evidence for force-generation by the Z-ring... 28
Modeling the Z-ring ... 29
Force-induced cell wall shaping... 31
Chapter 3: Differential bacterial surface display of peptides by the transmembrane domain of OmpA ...35
ABSTRACT... 35
INTRODUCTION... 36
MATERIALS AND METHODS... 38
RESULTS... 42
Design of loop insertions... 42
Growth of cells expressing OmpA-177 loop insertion proteins ... 44
Expression of OmpA-177 loop insertion proteins ... 46
Role of the periplasmic domain... 47
OM incorporation of truncate and full-length constructs ... 50
Surface display of loop insertions: fluorescent labeling of cells ... 52
DISCUSSION... 55
Contents
Reduced protein levels of FLAG or myc loop insertions in OmpA ... 55
Overexpression of engineered OmpA variants in LMC500 versus MC1061... 56
“Aberrant” heat-modifiability versus normal heat-modifiability ... 57
Summary ... 57
SUPPLEMENTARY MATERIALS AND METHODS... 62
Chapter 4: Domain fusions to the C-terminus of cell division protein FtsQ ...65
ABSTRACT... 65
INTRODUCTION... 66
RESULTS... 70
Detection of fusion proteins on immunoblot... 70
Localization and complementation of GFP-FtsQ and GFP-FtsQ-HSV ... 72
Localization of GFP-FtsQ-AcrA-X fusions in the presence of wild-type FtsQ... 76
Localization of GFP-FtsQ-AcrA-X fusions in the presence of FtsQ(E125K) ... 78
Extending GFP-FtsQ with a myc linker and the ALBP domain ... 80
DISCUSSION... 82
FtsQ as part of the divisome appears dynamic ... 82
Why are GFP-FtsQ-AcrA-X fusions excluded from mid-cell? ... 83
Recommendations for future work on GFP-FtsQ-myc-ALBP ... 85
MATERIALS AND METHODS... 87
Chapter 5: Outer membrane assembly of N- and C-terminal fusions to the OmpA transmembrane domain...93
ABSTRACT... 93
INTRODUCTION... 94
RESULTS... 98
A C-terminal Pal fusion to the OmpA TM domain... 98
(OmpA-177)-Pal in wild-type cells is excluded from mid-cell... 100
Localization of (OmpA-177)-Pal in ΔPal cells ... 102
A C-terminal mCherry fusion to the OmpA TM domain... 103
An N-terminal mCherry fusion to the OmpA TM domain ... 106
An N-terminal ALBP2 fusion to the OmpA TM domain ... 110
An N-terminal Pal-mCherry fusion to the OmpA TM domain... 111
An N-terminal TamiC fusion to the OmpA TM domain... 115
DISCUSSION... 116
OMP domain organization... 116
Toxicity of fusions... 117
Mid-cell exclusion of (OmpA-177)-Pal ... 118
Why the N-terminal Pal and TAmiC fusions to the OmpA TM domain are degraded: incompatibility with Lol and Tat systems respectively? ... 119
On export to the periplasm of heterologous proteins ... 120
MATERIALS AND METHODS... 122
Chapter 6: A counter-propagating optical tweezers setup with independent dual position detection ...131
ABSTRACT... 131
INTRODUCTION... 132
DETAILED EXPERIMENTAL SETUP AND CALIBRATION METHOD... 135
Optical tweezers setup... 135
Calibrating the optical trap ... 138
RESULTS... 139
Optical trapping at controlled temperatures... 139
Water objectives... 140
The roll-off frequency increases linearly with laser power... 142
DNA tether formation between beads in time-shared traps ... 143
Effect of laser irradiation on single growing cells ... 145
Summary ... 147
Chapter 7: Force-extension curves of DNA tethers attached to outer membrane protein OmpA in a living bacterium...149
ABSTRACT... 149
INTRODUCTION... 150
RESULTS... 154
DNA tethers to an immobilized bead ... 154
Axial dependence of the trap stiffness and trap center ... 159
Bacterial tethers to the OmpA β-barrel ... 165
Contents
What is the proper level of accuracy when analyzing bacterial tethers? ... 170
Bacterial F-x curves... 171
Bacterial tethers to full-length OmpA ... 175
Analysis of the measured unbinding forces at two pulling speeds... 178
DISCUSSION... 180
OmpA-177 versus full-length OmpA ... 180
Correcting for axial displacements... 182
Which unbinding force have we measured? ... 183
MATERIALS AND METHODS... 185
Chapter 8: Final Considerations and Recommendations ...191
Mobility of β-barrels in the OM ... 192
Restraining a β-barrel to mid-cell ... 194
Alternative approach: PG cell wall-less E. coli (“L-forms”) ... 196
Experimental geometry: assembly of the construct in the trap ... 197
Effect of laser light on bacterial growth... 199
Alternative geometries: surface and dumbbell approach ... 200
Increasing the strength of the protein-DNA connection... 203
Effect of forces on a growing bacterium ... 204
Concluding remarks... 205
Bibliography...207
Summary ...229
Samenvatting ...233
Dankwoord...239
Curriculum Vitae ...240
