A Framework to Assess Liquid‐Liquid‐Phase‐Separation in Bacterial Cells

Abstract

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.