Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria

Abstract

Subcellular phase separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates in Escherichia coli by utilizing electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates within E. coli cells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria. HighlightsO_LIMinimal disordered cationic peptides of varying charge densities can promote protein phase separation in bacterial cells. C_LIO_LIProtein net charge and charge-patterning are distinct determinants of phase behavior. C_LIO_LIProtein charge distribution can be used to tune the size, number, position, and reversibility of condensates. C_LIO_LIMultiple proteins can be selectively recruited to synthetic condensates with disordered cationic peptides. C_LI