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Title: The spatial and temporal characterisation of functional interactions between the key membrane stress proteins at the single molecule level in live Escherichia coli cells
Author: Mehta, Parul
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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All the cell types must maintain the integrity of their membranes which is important for cell viability. The membrane structure and function is maintained by many different response mechanisms. One such unique bacterial membrane stress response is the Phage shock protein response (Psp). It is composed of a transcriptional activator, negative regulator, signal sensors and transducers and stress effectors. Using milli second time-scale single-molecule fluorescence microscopy in live E. coli cells, the localisations, two dimensional diffusion dynamics and stoichiometry of functional Psp proteins were determined under non-stress and membrane stress conditions. The two major proteins studied in this research are a bacterial enhancer binding protein phage shock protein F (PspF) and the negative regulator and major effector phage shock protein A (PspA). For the imaging studies stable and functional chromosomal fusions of PspF and PspA to Venus fluorescent protein were used replacing the native proteins. It was established that a stable repressive PspF-PspA complex is located in the nucleoid and PspF displays DNA associated diffusion dynamics similar to other DNA-binding transcription factors such as LacI. PspF as a hexamer activated a single psp promoter at a time. The effector V-PspA assembled as higher order oligomers localised at the lateral membrane and showed very slow dynamics. In addition in vivo and in vitro structural studies of PspA showed that N-terminal amphipathic helix governed the balance between the dual functions of PspA. In a proposed model of the cellular landscape of the Psp response, the PspF-PspA inhibitory complex localised at the nucleoid transiently communicated with the polar regions of cells occupied by PspBC under non-stress conditions. With the stress conditions PspA retained at the polar membrane along with PspBC, while PspF associated with the active transcription complex to initiate the expression of psp genes. With the increase in the amounts of PspA, it organised itself into higher order effector moving along the MreB directed helical path contacting with the membrane via RodZ and in this way facilitated the organisation of membrane repair pathways at the damaged sites.
Supervisor: Buck, Martin; Ying, Liming Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available