Physicochemical properties driving membraneless organelle assembly in bacteria

项目摘要 最近，突破性的工作导致了一波生物分子凝聚油的发现。是这样的 将特定生物分子聚集在远离周围细胞环境的无膜细胞器中 长期以来一直是理论化的，现在实验上是容易驯服的。这些动态结构包含广泛的 蛋白质和核酸通过相分离的过程进行组装。虽然许多蛋白质是 倾向于相分离(单独或通过与其他蛋白质、核酸或小分子络合 分子)，这些凝聚体主要存在于真核细胞中。因为细菌通常不会 含有膜封闭的细胞器，我们假设细菌使用相分离 无膜细胞器作为细胞质的新组织者，调节生化活动 对不断变化的环境条件作出反应。 在这项提案中，我们的多学科团队将最先进的体外方法、体内实验、 并在计算机模拟和理论中探索细菌细胞质的结构组织和 细菌中相分离的无膜细胞器的特征。我们将重点研究一种候选蛋白质 系统，饥饿细胞(DPS)的DNA结合蛋白，驱动细菌的组织 染色体，并导致DNA在压力下在细菌细胞内形成一个单独的亚细胞室。我们 将首先研究该体系的化学和力学性质，绘制凝析油的相空间图 形成，确定它是通过旋节分解还是成核和凝析液滴发生的 生长，并测定其体外动力学。接下来，我们将阐明相分离如何控制访问 细胞质和类核相关的生物分子与细菌染色体和成像的结构 用活细菌中的无膜DNA组织细胞器来测量缩合对 体内的染色体结构和动力学。最后，我们将描述染色体阶段的影响 胞浆结合蛋白和DNA结合蛋白在体内的分离及其作用 染色体凝聚在细菌生理和生存中的作用。总而言之，我们的结果将定义 细菌细胞质独特的物理化学性质对分区化的贡献 在这些细胞内。相分离为中的空间和功能组织提供了另一种机制 生命中的细菌领域。事实上，阶段分离正在成为一项普遍的组织原则 生命之树，我们的工作最终将阐明生命的起源，并为理性地 设计了抗生素。

Project Summary Recently, breakthrough work has led to a wave of discoveries of biomolecular condensates. Such membraneless organelles that cluster specific biomolecules away from the surrounding cellular milieu have long been theorized and are now experimentally tractable. These dynamic structures contain a wide range of proteins and nucleic acids and assemble through the process of phase separation. While many proteins are prone to phase separation (either by themselves or via complexation with other proteins, nucleic acids, or small molecules), these condensates have primarily been found in eukaryotic cells. Since bacteria do not typically contain membrane-enclosed organelles, we hypothesize that bacteria instead use phase-separated membraneless organelles as novel organizers of their cytoplasm to regulate biochemical activity while they respond to changing environmental conditions. In this proposal, our multidisciplinary team combines state-of-the-art in vitro approaches, in vivo experiments, and in silico modeling and theory to explore the structural organization of the bacterial cytoplasm and characterize phase-separated membraneless organelles in bacteria. We will focus on a candidate protein system, the DNA-binding protein from starved cells (Dps), that drives the organization of the bacterial chromosome and leads DNA to form a separate subcellular compartment within bacterial cells upon stress. We will first study this system’s chemical and mechanical properties, map the phase space for condensate formation, ascertain whether it occurs through spinodal decomposition or nucleation and condensate droplet growth, and determine its kinetics in vitro. Next, we will elucidate how phase separation controls the access of cytoplasmic and nucleoid-associated biomolecules to the bacterial chromosome and image the structure of membraneless DNA-organizing organelles in living bacteria to measure the effect of condensation on chromosome structure and dynamics in vivo. Finally, we will characterize the impact of chromosome phase separation on the mobility of cytoplasmic and DNA-binding proteins in vivo and determine the role of chromosomal condensation in bacterial physiology and survival. Together, our results will define the contributions of the unique physicochemical properties of the bacterial cytoplasm to compartmentalization within these cells. Phase separation provides an alternate mechanism for spatial and functional organization in the bacterial domain of life. Indeed, phase separation is emerging as a universal organizing principle across the tree of life, and our work will ultimately shed light on the origin of life and provide new targets for rationally designed antibiotics.