Phytoplankton are microscopic plants that form the base of ocean food chains and collectively do half of the photosynthesis on Earth, equal to all the plants on land. Prochlorococcus is the single most abundant phytoplankton species in the global oceans and is responsible for producing an estimated 4 billion tons of fixed carbon each year, which is approximately the same productivity as global croplands. In addition they are the smallest and most efficient photosynthesizers designed by nature, relying only on sunlight, CO2 and essential nutrients for growth. For the past 25 years, research on this minimal prototroph has led to a growing understanding of its features across all scales of biological organization, from the genome to the global ecosystem. This fascinating microbe has also offered a great opportunity to foster public interest on topics such as ocean ecology, biodiversity and genomics. It attracts the attention of science journalists, and serves as an "ambassador" for microorganisms - exemplifying their important role in planetary maintenance. The one thing that is missing from the Prochlorococcus model system tool kit is the ability to do genetics. This bacterium has resisted all genetic manipulation attempts to date, which has significantly hampered the rate of discovery. The goal of this project is to directly address this limitation by developing an efficient genetic system for Prochlorococcus using both traditional and state-of-the-art tools developed specifically for this project.
Research on Prochlorococcus has expanded steadily over the past 25 years, as more and more strains have been brought into culture, and their distribution in the global oceans has been mapped extensively. The PIs now have 100s of Prochlorococcus cultures in the lab, an extensive amount of transcriptomics and proteomics data, and they continue to accumulate genomic data from cultures and wild populations. However each new Prochlorococcus strain, or single cell sequenced, adds roughly 100 entirely new genes to the Prochlorococcus pangenome, the vast majority of which are of unknown function. The development of a genetic system is necessary for identifying the functions of these unknown genes and testing hypotheses involving those with putative functions. This project notably involves a high throughput microfluidic electroporation technology to transform recalcitrant microbes orders of magnitude faster than traditional approaches. A global mutagenesis (Transposon-based tools) and targeted mutagenesis (CRISPR-based tools) strategy will be implemented for Prochlorococcus, while optimizing the microfluidic electroporation technology to its full potential. As proof-of-concept, each tool will be tested in its ability to efficiently reveal gene functions by using our current known repertoire of nutrient acquisition genes in Prochlorococcus.