Freshwater picocyanobacteria are an essential component of our waterbodies, fulfilling a role of global primary producers in waters which are often limited or co-limited by nitrogen. However, they remain relatively poorly understood, especially in comparison to their marine relatives. Also poorly understood is the picocyanobacterial metabolism of organic sources of nitrogen. Researchhas traditionally focused on inorganic sources such as ammonium or nitrate, however it is becoming increasingly evident that organic nitrogen is bioavailable and an integral component of the freshwater nitrogen pool. In this thesis,we sequence five strains of freshwater picocyanobacteria and compare theirencoding capabilities to a common Synechococcus model cyanobacterium (Synechococcus elongatus). We increased the representation of freshwater picocyanobacteria, and found reduced encoding of nitrite-associatedassimilation genes in picocyanobacteria. Further differences in encoded antennae proteins highlight the differences between freshwater picocyanobacteria (Synechococcus spp. of the Syn/Proclade) and Synechococcus elongatus, highlighting the need for a ’true’ freshwater picocyanobacterium model organism. Utilising comparative genomic analyses we investigated the nitrogen assimilation capabilities of freshwater picocyanobacteria, comparing these to picocyanobacteria of different habitatsin addition to larger freshwater cyanobacteria. The diversity displayed among nitrogen assimilation capabilities reveals the evolutionary history of the picocyanobacteria, showcasing the environments in which these organisms evolved. These analyses revealed significant variation in the encoded aminoacid transporters between freshwater picocyanobacteria and larger freshwater cyanobacteria. With different amino acid transporters having different amino acid preferences and uptake rates, this may have implications for amino acid bioavailability. Other sources of potentially differentially bioavailable nitrogen include novel organic forms, such as chitin and glyphosate. Finally, we conduct a growth assay to determine the assimilation capabilities of amino acids as an organic nitrogen source, and explore the proteomic response to growth on selected amino acids using quantitative proteomic analysis. The growth assay revealed widespread amino acid assimilation as a sole nitrogen source for freshwater picocyanobacteria, enabling a wide N pool which may contribute to their limnetic dominance. However, proteomic analysis revealed a subtle stress response in freshwater picocyanobacteria when grown on selected amino acids, potentially due to the accumulation of metabolites. Together, the work increasesour understanding of the nitrogen assimilation capabilities of freshwater picocyanobacteria, with a focus on organic sources of nitrogen
