Single-Molecule Tracking of RNA Polymerase In and Out of Condensates in Live Bacterial Cells.

Biomolecular condensates, first discovered in eukaryotic cells, were recently found in bacteria. The small size of these organisms presents unique challenges for identifying and characterizing condensates. Here, we describe a single-molecule approach for studying biomolecular condensates in live bacterial cells.

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation.

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media.