Formation of Biomolecular Condensates in Bacteria by Tuning Protein Electrostatics

While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates-organelles that lack a membrane-provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid-liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in E. coli. Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.