AbstractThis thesis tells the story of my doctoral research in chronological order; a story that is divided into four acts.
The first act is the prologue, which narrates my exploration into the world of two-component signalling—a system familiar amongst bacteria, but one that gradually disappears further down the phylogenetic tree, which is the main reason that this system would be an excellent novel antibiotic target. Dubbed as the canonical two-component system (TCS) due to it being the most well-understood of its kind, EnvZ/OmpR still raises questions regarding several aspects of its signalling mechanism, one of which regards its transmembrane domain, and that is empirically addressed in this thesis.
The second act addresses the interaction between the transmembrane helices of EnvZ and its correlation to signal output quantified by ompC and ompF transcription. Using sulfhydryl-reactivity experiments, our studies showed that the most prominent structural shifts occurred at the second transmembrane domain, following a non-piston model of movement, whereas shifting in the first transmembrane domain mainly occurred near the periplasmic region, implying possible interaction with other periplasmic protein controlling EnvZ sensory mechanism. Results presented here provide insight into the question of what role the transmembrane domains play in the signal transmission process within sensor histidine kinases.
The third act addresses my attempt to create a chimeric receptor by fusing the sensory region of S. Typhimurium magnesium sensor, PhoQ, with the catalytic region of a well-characterised EnvZ. Results showed that simply inserting the gene into our backbone, pRD400, and aromatically tuning the receptor were not enough to completely restore the sensory function of the PhoQ-native domain. A different approach to plasmid design was undertaken using the Standard European Vector Architecture (SEVA) platform, which allowed for better control of receptor expression as demonstrated by immunoblotting results. Further analysis on signal output still needs to be performed to assess if expression of the chimeric receptor using the SEVA backbone could restore the sensory function, and produce a dose-response signal output, which would be the ideal signal pattern required if this chimera were to be used in a high-throughput screening platform.
The fourth and final act concludes the story of transmembrane signalling and chimera design, and furthermore lays out plans on how the projects are or could be expanded within the research group, particularly which questions still need to be answered: the nature of interdomain interaction during EnvZ signalling, and how to optimise the design of the PhoQ-EnvZ chimera.
|Date of Award||Jan 2019|
|Supervisor||Roger Draheim (Supervisor) & Ivor Ebenezer (Supervisor)|