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- PublicationProteomics Approaches for the analysis of polyhydroxyalkanoate production in Pseudomonas putida KT2440(University College Dublin. School of Biomolecular and Biomedical Science, 2022)Polyhydroxyalkanoates (PHAs) are biodegradable polymers which can be used a starting material for plastic, offering an alternative to petrochemical based plastic. PHA is produced by many bacteria as a form of carbon storage and is stored in a granule composed of a polymer core, surrounded by a layer of proteins known as granule-associated proteins (GAPs). A better understanding of PHA production is critical for their use as a bioplastic. In this thesis, modern proteomic approaches were used to investigate PHA production in Pseudomonas putida KT2440. A combination of three approaches were used to study PHA production: expression proteomics, subcellular fractionation proteomics and protein interaction studies. While many studies have focused on specific enzymes involved in the production of PHA, the changes that occur on a whole proteome level are less understood. Therefore, we firstly implemented a liquid chromatography mass spectrometry (LCMS) approach to gain an insight into proteome status at various timepoints in both PHA accumulating and PHA non-accumulating conditions, using glucose as a carbon source. Using this approach, 52% of the theoretical P.putida proteome was successfully analysed. Statistical analysis revealed that proteins mapping to key pathways such as nitrogen metabolism and ABC transporters showed coordinated expression changes. Proteins involved in the PHA production pathway were also upregulated in response to PHA accumulating conditions, including PhaI and PhaF, which play an important role in PHA granule formation and segregation. Secondly, subcellular fractionation proteomics was employed to isolate pure PHA granules from P.putida and the resulting preparation analysed by LCMS to determine the protein composition. Over 1,000 proteins were identified, requiring data analysis and validation steps to prioritise the most likely GAPs. All 6 proteins expressed from the PHA operon were identified in the granule proteome. Other proteins were evaluated based on factors including a) enrichment on the granule b) up-regulation under conditions known to induce granule formation c) reports from the scientific literature d) biophysical factors predicted using bioinformatics. As well as confirming previous descriptions of the PHA granule proteome, my work expanded the set of potential granule proteins, including 87 proteins that were enriched on the PHA granule. Thirdly, protein interaction studies were carried, focused on proteins involved in PHA synthesis. Few protein interaction studies have been carried out on PHA-related proteins, and little is known about how these proteins interact with each other. I implemented a fusion protein strategy, fusing enhanced yellow fluorescent protein (eYFP) to the C-terminal of five proteins that were known to be involved in PHA production: Crc global regulator, PhaI, PhaD PhaZ and PhaC1. Fusion proteins were subjected to affinity purification mass spectrometry (APMS) to identify potential interactors. I constructed a database of the results, supplemented with information describing the bait and prey proteins. Only one previously identified interaction between PHA pathway components was observed, between PhaI and PhaF. 79 potential interactions were observed with significance and evaluated using additional criteria. 3 proteins identified were selected for further validation studies: pyruvate dehydrogenase subunit E1 (Q88QZ5), putative lipoprotein (Q88F99) and peptidoglycan-associated lipoprotein (P0A138). Reverse pulldown and microscopy studies were used to validate protein interactions and confirm subcellular localisation. We confirmed the interaction of P0A138 and PhaF and the localisation of P0A138 to the PHA granule in vivo. We also confirmed the interaction between Q88QZ5 and PhaF and that Q88QZ5 localises in vivo to the PHA granule. This finding reveals a potential link between PHA granule production during stress conditions and central metabolism.