CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote

Despite their importance to aquatic ecosystems, global carbon cycling, and sustainable bioindustries, the genomes of photosynthetic bacteria contain large numbers of uncharacterized genes. Here, we develop high-throughput endogenous fluorescent protein tagging in the cyanobacterium <i>Synechococcus elongatus</i> PCC 7942. From 400 targets, we successfully tag over 330 proteins corresponding to >10% of the proteome. We use this collection to determine subcellular localization, relative protein abundances, and protein-protein interaction networks, providing biological insights into diverse processes-from photosynthesis to cell division. We build a high-confidence protein-protein interaction map for the major components of photosynthesis, associating previously uncharacterized proteins with different complexes and processes. In response to light changes, we visualize, on second timescales, the reversible formation, growth, and fusion of puncta by two Calvin cycle proteins, suggesting that biomolecular condensation provides spatiotemporal control of the Calvin cycle in cyanobacteria. We envision that these insights, cell lines, and optimized methods will facilitate rapid advances in cyanobacteria biology and, more broadly, all photosynthetic life.