Single-molecule imaging of electroporated chemotaxis proteins in live bacteria
<p>Many species of motile bacteria use rotating extracellular filaments to propel themselves through liquid media. Each filament is driven by a membrane spanning rotary nano-machine called the bacterial flagellar motor. In <em>Escherichia coli</em> and <em>Rhodobacter sphaero...
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| Format: | Thesis |
| Language: | English |
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2015
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| _version_ | 1826280549931548672 |
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| author | Di Paolo, D |
| author2 | Berry, R |
| author_facet | Berry, R Di Paolo, D |
| author_sort | Di Paolo, D |
| collection | OXFORD |
| description | <p>Many species of motile bacteria use rotating extracellular filaments to propel themselves through liquid media. Each filament is driven by a membrane spanning rotary nano-machine called the bacterial flagellar motor. In <em>Escherichia coli</em> and <em>Rhodobacter sphaeroides</em> the motor is powered by a transmembrane flux of H+ and the chemical energy is converted into work through a ring of stator units pushing on a central rotor.</p> <p><em>Chemotaxis</em> is the biasing of movement towards regions that contain higher concentrations of beneficial, or lower concentrations of toxic, chemicals and is one of the most well-understood bacterial sensory pathways. Upon phosphorylation, the response regulator protein CheY transduces changes of environmental chemical gradients detected by specific transmembrane chemoreceptors to the flagellar motors: it binds to the N-terminus of the FliM proteins in the C-ring part of the motor inducing a cascade of conformational changes that modulate the direction of rotation (in <em>E. coli</em>) or the motor stopping (in <em>R. sphaeroides</em>).</p> <p>In this project, a novel technique for protein internalisation in live bacteria based on electroporation and single-molecule imaging using a custom-built microscope are combined to perform an in-depth investigation of the interactions between wild type and mutant chemotaxis proteins, chemoreceptors and the motor complex <em>in vivo</em>.</p> <p>Chemotaxis proteins are purified, labelled with organic dyes and inserted into live <em>E.coli</em> and <em>R. sphaeroides</em> cells by electroporation. In typical experiments exploiting this new capability, video fluorescence microscopy shows single molecules diffusing within cells, interacting with the sensory clusters and individual flagellar motors. The work described in this thesis allows for the first time imaging and tracking of single dye-labelled chemotaxis proteins performing their function as response regulators in real time. Diffusion as well as relevant binding constants and dwell times at each end of their journey are measured, providing also a comparison of such quantities across different protein mutants, genetic backgrounds and environmental conditions.</p> |
| first_indexed | 2024-03-07T00:15:22Z |
| format | Thesis |
| id | oxford-uuid:7aa5fabc-2237-480f-8615-5225f009df82 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:15:22Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | oxford-uuid:7aa5fabc-2237-480f-8615-5225f009df822022-03-26T20:45:26ZSingle-molecule imaging of electroporated chemotaxis proteins in live bacteriaThesishttp://purl.org/coar/resource_type/c_db06uuid:7aa5fabc-2237-480f-8615-5225f009df82Molecular biologyBiochemistryBiophysicsEnglishORA Deposit2015Di Paolo, DBerry, R<p>Many species of motile bacteria use rotating extracellular filaments to propel themselves through liquid media. Each filament is driven by a membrane spanning rotary nano-machine called the bacterial flagellar motor. In <em>Escherichia coli</em> and <em>Rhodobacter sphaeroides</em> the motor is powered by a transmembrane flux of H+ and the chemical energy is converted into work through a ring of stator units pushing on a central rotor.</p> <p><em>Chemotaxis</em> is the biasing of movement towards regions that contain higher concentrations of beneficial, or lower concentrations of toxic, chemicals and is one of the most well-understood bacterial sensory pathways. Upon phosphorylation, the response regulator protein CheY transduces changes of environmental chemical gradients detected by specific transmembrane chemoreceptors to the flagellar motors: it binds to the N-terminus of the FliM proteins in the C-ring part of the motor inducing a cascade of conformational changes that modulate the direction of rotation (in <em>E. coli</em>) or the motor stopping (in <em>R. sphaeroides</em>).</p> <p>In this project, a novel technique for protein internalisation in live bacteria based on electroporation and single-molecule imaging using a custom-built microscope are combined to perform an in-depth investigation of the interactions between wild type and mutant chemotaxis proteins, chemoreceptors and the motor complex <em>in vivo</em>.</p> <p>Chemotaxis proteins are purified, labelled with organic dyes and inserted into live <em>E.coli</em> and <em>R. sphaeroides</em> cells by electroporation. In typical experiments exploiting this new capability, video fluorescence microscopy shows single molecules diffusing within cells, interacting with the sensory clusters and individual flagellar motors. The work described in this thesis allows for the first time imaging and tracking of single dye-labelled chemotaxis proteins performing their function as response regulators in real time. Diffusion as well as relevant binding constants and dwell times at each end of their journey are measured, providing also a comparison of such quantities across different protein mutants, genetic backgrounds and environmental conditions.</p> |
| spellingShingle | Molecular biology Biochemistry Biophysics Di Paolo, D Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title | Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title_full | Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title_fullStr | Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title_full_unstemmed | Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title_short | Single-molecule imaging of electroporated chemotaxis proteins in live bacteria |
| title_sort | single molecule imaging of electroporated chemotaxis proteins in live bacteria |
| topic | Molecular biology Biochemistry Biophysics |
| work_keys_str_mv | AT dipaolod singlemoleculeimagingofelectroporatedchemotaxisproteinsinlivebacteria |