Summary of Parent Grant Recently, breakthrough work has led to a wave of discoveries of biomolecular condensates. Such membraneless organelles that cluster specific biomolecules away from the surrounding cellular milieu have long been theorized and are now experimentally tractable. These dynamic structures contain a wide range of proteins and nucleic acids and assemble through the process of phase separation. While many proteins are prone to phase separation (either by themselves or via complexation with other proteins, nucleic acids, or small molecules), these condensates have primarily been found in eukaryotic cells. Since bacteria do not typically contain membrane-enclosed organelles, we hypothesize that bacteria instead use phase-separated membraneless organelles as novel organizers of their cytoplasm to regulate biochemical activity while they respond to changing environmental conditions. In this proposal, our multidisciplinary team combines state-of-the-art in vitro approaches, in vivo experiments, and in silico modeling and theory to explore the structural organization of the bacterial cytoplasm and characterize phase-separated membraneless organelles in bacteria. We will focus on a candidate protein system, the DNA-binding protein from starved cells (Dps), that drives the organization of the bacterial chromosome and leads DNA to form a separate subcellular compartment within bacterial cells upon stress. We will first study this system’s chemical and mechanical properties, map the phase space for condensate formation, ascertain whether it occurs through spinodal decomposition or nucleation and condensate droplet growth, and determine its kinetics in vitro. Next, we will elucidate how phase separation controls the access of cytoplasmic and nucleoid-associated biomolecules to the bacterial chromosome and image the structure of membraneless DNA-organizing organelles in living bacteria to measure the effect of condensation on chromosome structure and dynamics in vivo. Finally, we will characterize the impact of chromosome phase separation on the mobility of cytoplasmic and DNA-binding proteins in vivo and determine the role of chromosomal condensation in bacterial physiology and survival. Together, our results will define the contributions of the unique physicochemical properties of the bacterial cytoplasm to compartmentalization within these cells. Phase separation provides an alternate mechanism for spatial and functional organization in the bacterial domain of life. Indeed, phase separation is emerging as a universal organizing principle across the tree of life, and our work will ultimately shed light on the origin of life and provide new targets for rationally designed antibiotics.