Abstract

PHB granules are bacterial organelles that store excess carbohydrates in the form of water-insoluble polyhydroxybutyrate (PHB). The PHB polymerase, phasin (a small amphipathic protein), and active PHB synthesis are essential for the formation of mature PHB granules in Caulobacter crescentus. Granule formation was found to be initiated by the condensation of self-associating PHB polymerase-GFP into foci, closely followed by the recruitment and condensation of phasin-mCherry. Following the active synthesis of PHB and granule maturation, the polymerase dissociates from mature granules and the PHB depolymerase is recruited to the granule. The polymerase directly binds phasin in vitro through its intrinsically disordered N-terminal domain. Thus, granule biogenesis is initiated and controlled by the action of a PHB polymerase and an associated helper protein, phasin, that together synthesize the hydrophobic granule content while forming the granules protein boundary. ImportanceLike eukaryotes, bacteria organize their cytoplasm in subcellular compartments. These bacterial compartments can be membrane-bound (e.g. magnetosomes), or membraneless with protein-encased shells (e.g. carboxysomes). Here we investigate how Caulobacter forms membrane-less compartments that store the water-insoluble carbohydrate polymer polyhydroxybutyrate (PHB). A PHB polymerase is essential for granule biogenesis and we observed a direct interaction with the granule associated protein phasin through the disordered N-terminus of the polymerase. We found that PHB granules form by sequential recruitment of key proteins, beginning with the polymerase, and that the granule composition changes as these organelles mature.