Mostyn Brown Thesis 2009

Name: Mostyn Brown Thesis 2009
Published: 2011-01-01T00:00:00Z
License: https://ora.ox.ac.uk/terms_of_use

Brown, M

Documentation:: This dataset is a part of the project work referenced by the thesis titled CONTROL OF THE UNIDIRECTIONAL MOTOR IN RHODOBACTER SPHAEROIDES by Mostyn T. Brown submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Oxford The dataset contains the following: * The LATEX files including the style file and figures used to compile the thesis * Videos * Protocols used * Bead Rotation Data divided into the following sections (1) Wild-type Bead Rotation Data, (2) JPA1353 Bead Rotation Data, (3) Flow Cell Bead Data, and (4) Analysis Programs

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APA Style

Brown, M. (2011). Mostyn Brown Thesis 2009. University of Oxford.

MLA Style

Brown, M. Mostyn Brown Thesis 2009. University of Oxford, 2011.

Chicago Style

Brown, M. 2011. Mostyn Brown Thesis 2009. University of Oxford.
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Files:: Mostyn_thesis_template.zip

(zip, 1.5MB, Terms of use)

protocols.zip

(zip, 5.0MB, Terms of use)

rawdata.zip

(zip, 251.3MB, Terms of use)

thesis.pdf

(Preview, pdf, 12.3MB, Terms of use)

thesis.zip

(zip, 233.1MB, Terms of use)

videos.zip

(zip, 3.8MB, Terms of use)

Authors/Creators

+ Brown, M More by this author/creator

Institution:: University of Oxford
Division:: MSD
Department:: Biochemistry
Role:: Creator

+ Biotechnology and Biological Sciences Research Council More from this funder

Publisher:: University of Oxford
Publication date:: 2011
DOI:: 10.5287/bodleianFdk5.3
Data collected:: 2005-10-01 - 2008-10-01

Language:: English
Keywords:: clutch

flagellum

brake

flagellar motor

chemotaxis

rhodobacter
Pubs id:: 1480884
UUID:: uuid:7a012390-cb5d-42ad-a916-897d27c034b0
Local pid:: pubs:1480884
Deposit date:: 2015-07-23
ARK identifier:: ark:/29072/ora_7a012390cb5d42ada916897d27c034b0

Related item:: Control of the unidirectional motor in Rhodobacter sphaeroides
Description:: The control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques.
High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively.
Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively).
To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad).
Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units.

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