A study into the properties and applications of bacterial microcompartments and polyphosphate metabolism

Abstract

Bacterial microcompartments were initially observed in the 1950s and since that time, research in the field has focused on the unique properties of their proteinaceous structures and their role in the bacterial cell. This thesis investigates the properties of the propanediol utilization (Pdu) and ethanolamine (Eut) utilization microcompartments and applications of their components in the cells of Lactobacillus reuteri, and Escherichia coli predominantly, with additional investigation into Yersinia spp. The experimental chapters of this thesis initially investigate the application of bacterial microcompartments as tools for compartmentalizing metabolic reactions, in particular regarding the uptake of phosphate from extracellular environment and the retention of polyphosphate intracellularly. This was observed experimentally through the engineering of recombinant constructs containing genes encoding microcompartment-directed polyphosphate kinase enzyme. Bacterial phenotypes conferred by these constructs with respect to enhanced net uptake of phosphate from the environment were defined, and potential applications as a clinical therapy addressed in an animal trial, which utilized the phosphate-uptake constructs which had been created. In the trial, recombinant bacteria expressing these constructs were successfully delivered in therapeutic quantities to the target (small intestine) of rats experiencing induced renal failure, with no adverse effects were observed. However inter-group animal variation regarding baseline blood mineral content was larger than the expected therapeutic effect and therefore no therapeutic effect could be demonstrated. Additionally, novel phenotypic differences conferred by induction of Pdu and Eut microcompartments and empty recombinant microcompartments on host cells were identified. These identified an observable phenotypic difference in buoyant density between strains expressing the Pdu and Eut microcompartments. Further investigation into the potential function and phenotypic differences of strains expressing microcompartments were supported by methodological developments including the use of imaging instruments such as the FIB-SEM, and microcompartment extraction from cells. These technical developments enabled additional observations regarding the characteristics of microcompartments and provided novel information.