Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.714011
Title: Single-molecule imaging of electroporated chemotaxis proteins in live bacteria
Author: Di Paolo, Diana
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Abstract:
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 Escherichia coli and Rhodobacter sphaeroides 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. Chemotaxis 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 E. coli) or the motor stopping (in R. sphaeroides). 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 in vivo. Chemotaxis proteins are purified, labelled with organic dyes and inserted into live E.coli and R. sphaeroides 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.
Supervisor: Berry, Richard Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.714011  DOI: Not available
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