Physicochemical properties driving membraneless organelle assembly in bacteria

Project Summary
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.

Grant Number: 5R01GM143182-04
NIH Institute/Center: NIH
Principal Investigator: Julie Biteen