Biophysical characterisation of the bacterial chemosensory protein, PscF
|dc.contributor.advisor||Gerth, Monica Leigh|
|dc.contributor.author||Boult, Sean Alexander Graham|
|dc.identifier.citation||Boult, S. A. G. (2019). Biophysical characterisation of the bacterial chemosensory protein, PscF (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/9128||en|
|dc.description.abstract||In bacteria, movement in response to a chemical stimulus is known as chemotaxis. Chemotaxis is controlled by a class of ligand binding proteins called chemoreceptors and works to bias cell movement towards environments that are favourable for growth and survival. This phenomenon and the proteins involved have been linked to competitive fitness and virulence in various model organisms. However, outside of enteric bacteria, the specific chemotactic signals that pathogens respond to are not particularly well understood. To help address this gap in knowledge, this study sought to screen the chemical-sensing profiles of novel chemoreceptors belonging to the kiwifruit pathogen, Pseudomonas syringae pv. actinidiae (Psa). The genome of Psa strain NZ-V13 is predicted to encode 43 chemoreceptors. For the vast majority, neither what they detect nor how they detect it is understood. For the work described herein, three Psa chemoreceptors (locus IDs Psa_00575, Psa_25325 and Psa_26265), each representing a major structural-fold family, were selected for investigation. First, the ligand binding domains (LBDs) of Psa_00575, Psa_25325 and Psa_26253, were cloned and expressed. Based on sequence analysis and protein purification trials, Psa_00575 was selected for further characterisation (henceforth referred to as PscF). Using a high-throughput fluorescence-based thermal shift assay, the LBD of PscF was screened against a library of ~500 potential ligands. Results from this high-throughput screening identified a single candidate ligand, formate. PscF-formate binding was subsequently investigated using isothermal titration calorimetry and X-ray crystallography. Finally, formate chemotaxis in Psa was confirmed in vivo using chemotaxis assays and preliminary evidence was obtained from growth curves that suggest environmental formate increases the growth rate of Psa inoculants. Not only does this work contribute to our understanding of molecular recognition in chemoreceptors, but it may also help us better understand their role in host invasion and colonisation.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Biophysical characterisation of the bacterial chemosensory protein, PscF|
|thesis.degree.name||Master of Science|
|thesis.degree.grantor||University of Otago|
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