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 Escherichia coli. 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. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform 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 E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

The Type I Interferon family of cytokines all act through the same cell surface receptor and induce phosphorylation of the same subset of response regulators of the STAT family. Despite their shared receptor, different Type I Interferons have different functions during immune response to infection. In particular, they differ in the potency of their induced anti-viral and anti-proliferative responses in target cells. It remains not fully understood how these functional differences can arise in a ligand-specific manner both at the level of STAT phosphorylation and the downstream function. We use a minimal computational model of Type I Interferon signaling, focusing on Interferon-α and Interferon-β. We validate the model with quantitative experimental data to identify the key determinants of specificity and functional plasticity in Type I Interferon signaling. We investigate different mechanisms of signal discrimination, and how multiple system components such as binding affinity, receptor expression levels and their variability, receptor internalization, short-term negative feedback by SOCS1 protein, and differential receptor expression play together to ensure ligand specificity on the level of STAT phosphorylation. Based on these results, we propose phenomenological functional mappings from STAT activation to downstream anti-viral and anti-proliferative activity to investigate differential signal processing steps downstream of STAT phosphorylation. We find that the negative feedback by the protein USP18, which enhances differences in signaling between Interferons via ligand-dependent refractoriness, can give rise to functional plasticity in Interferon-α and Interferon-β signaling, and explore other factors that control functional plasticity. Beyond Type I Interferon signaling, our results have a broad applicability to questions of signaling specificity and functional plasticity in signaling systems with multiple ligands acting through a bottleneck of a small number of shared receptors.

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