Department of Biology, McGill University, Montreal, QC H3A 1B1, Canada.
Department of Physics, McGill University, Montreal, QC H3A 2T8, Canada.
Proc Natl Acad Sci U S A. 2020 Aug 4;117(31):18540-18549. doi: 10.1073/pnas.2005019117. Epub 2020 Jul 16.
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. 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.
细菌细胞曾经被描述为仅仅是“酶袋”，但实际上它们高度组织化，许多大分子表现出不均匀的定位模式。然而，控制这种空间异质性的物理和生化机制在很大程度上仍然未知。在这里，我们确定液-液相分离（LLPS）是在富含营养的培养基中，细胞进入对数生长期时，RNA 聚合酶（RNAP）快速从弥散状态转变为聚集状态。RNAP 聚集体对己二醇敏感，己二醇是一种能溶解真核细胞中类似液体的隔室的化学物质。此外，我们发现转录终止因子 NusA 在体外和体内形成液滴，这表明它可能是 RNAP 聚集体的核。最后，我们使用单分子追踪来描述聚集体成分的动力学。我们的结果表明，RNAP 和 NusA 分子在聚集体内部移动，其迁移率比 DNA 位点数，但比核质通过核的整体扩散慢。我们得出结论，RNAP 聚集体是通过 LLPS 组装而成的生物分子凝聚物。这项工作为细菌中的 LLPS 提供了直接证据，并表明该过程可以作为原核生物和真核生物细胞内组织的一种机制。
