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Structural studies of the FliG protein of the bacterial flagellar rotor
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Tupiņa, Dagnija (2022) Structural studies of the FliG protein of the bacterial flagellar rotor. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3903273
Abstract
Flagella are complex multiprotein structures that not only enable bacteria to move through the environment, but are also key virulence factors for pathogens. The flagellar rotor that drives the entire flagellum, with rotation that can happen in both directions, is composed of the C-ring (built of proteins FliG, FliM and FliN) and MS-ring (built of the FliF protein). Interaction between FliG and FliF proteins and their higher-order complex formation is central to the bacterial flagellum biosynthesis. This is because FliF and its co-folding partners are among the earliest structures to assemble during the flagella building process, and because they physically link all the flagellar associated structures in the cytoplasm with those in the periplasmic space (in Gram negative bacteria), outer-membrane and finally the filament that resides outside the cell. Meanwhile, FliG functions to transfer the torque from the membrane anchored stators, transducing the energy from ion flow to rotation of the rotor and thus the entire flagellar structure. To expand our mechanistic understanding of this molecular machine, a combination of methods was employed here. Molecular dynamics simulations were used to study the structure of the FliG, revealing that the linker between the FliG N-terminal and middle domains likely adopts an extended conformation in vivo, in contrast with crystallographic data. An integrative modelling approach was then taken, encompassing homology modelling and molecular dynamics flexible fitting approaches, to create full length viable FliG models in a C-ring assembly. Furthermore, no high resolution structure of the FliG ring has been solved to date; thus, two cryo-electron tomography datasets were collected to visualize motor reconstructions from C. jejuni (a pathogen and a flagella research model organism), and preliminary reconstructions were obtained. Construction of a ΔfliF strain and a set of complementation strains was created to study a function of previously identified pseudorevertant mutations in FliF that restored motility to non-motile C. jejuni. Whole genome sequencing and negative stain electron microscopy highlighted the importance of the two genes coding FliG and FliF to be co-transcribed and co-translated for an efficient motor assembly. Sequence analysis also identified a novel mutation in FliF associated with increased motility. Collectively, these methods expanded the knowledge of the bacterial flagellar rotor, as well as providing models to generate new hypotheses that lay groundwork for future experiments.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH301 Biology Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Flagella (Microbiology), Biosynthesis, Microorganisms -- Motility, Cryoelectronics, Electron microscopy, Homology (Biology), Campylobacter jejuni | ||||
Official Date: | September 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Constantinidou, Chrystala ; Bond, Peter John | ||||
Format of File: | |||||
Extent: | 199 pages : colour illustrations, charts | ||||
Language: | eng |
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