Polypeptide (dis)assembly in bacterial spores and microalgae

Abstract

The ability to form an endospore allows bacteria of certain genera to survive unfavourable environmental conditions. This durability is known to be related to the properties of the concentric protein shells that surround the spore, forming a distinct layered coat. This thesis begins with experimental work to characterise the proteins in the bacterial spore coat and cytoskeleton of microalgae. Using early versions of AlphaFold2 that were released during the course of the work, the thesis goes on to add predictions of protein structure to form insights based on information at the molecular level. A comprehensive atlas of the structures of each coat protein was created, starting with the model species Bacillus subtilis and extending to other species with certain outer layers, such as the exosporium, that are not present in B. subtilis. It is the interaction between different proteins that gives the bacterial spore coat its properties, necessitating the use of AlphaFold-multimer to evaluate the presence of protein complexes. An example is the six proteins in the GerP operon that moderate the response of a spore to factors that trigger germination. Another important complex to consider contains the proteins involved in the sporulation process that have evidence of interaction based on experimental observations reported in the literature. These include pentameric homo-oligomers which contain a symmetrical assembly of the same subunit, and hetero-oligomers such as the germinant receptors GerAA, GerAB and GerAC, with one copy of each, forming a complex with the proteins tightly nestled together at a distance small enough for interaction to occur. Knowing that these interactions occur, novel designs of proteins can be made with a defined length but no fixed amino acid residue identity, and these can be set to bind to target proteins and iterated until close contacts between the binder and target are predicted. The thesis has been organised into seven chapters: Chapter 1 introduces the thesis starting with polypeptide properties and classifications, looking at methods of determining protein structure and their importance in the properties of bacterial spores. The aims and objectives of the thesis are contextualised with a literature review, identifying research gaps and explaining how the rest of the thesis is structured. Chapter 2 presents the experimental work undertaken on bacterial spores to modify coat proteins with fluorescent tags and use microscopy to quantify the coat layer location. Chapter 3 presents the experimental work on microalgae where controlling growth conditions and investigating methods to break down the cytoskeleton are undertaken. It considers the disassembly of proteins in microalgae and the potential uses of this process in biotechnology for manufacturing biodiesel. Chapter 4 shows the impact of theoretical investigations using AlphaFold to predict the structures of the proteins considered in the previous chapters, providing a basis for interactions based on information at the molecular level to understand why the experimental observations occur. Chapter 5 considers the combination of proteins that form complexes, both as copies of the same protein and between different proteins that are known to interact or are translated together. Chapter 6 evaluates novel design of protein binders that target coat proteins, offering a solution to disrupt the structure of the spore coat by interfering with the interactions that bind the coat proteins together, for potential application in decontamination and sterilisation processes. Chapter 7 delivers the conclusions to this thesis and provides perspectives for what can be done with later developments to the methods used in this thesis.