Structural and Dynamical Response of Escherichia coli to Osmotic Stress

这一部分与最初的提案相比只有轻微的变化。 细菌在自然界中无处不在，证明了它们具有极强的适应性。大肠杆菌必须 通常以温度变化、营养物质等形式对外界压力做出反应 缺乏，药物的存在，或外部溶质浓度的变化(渗透 压力)。在这个项目中，我们研究了蛋白质的运输(扩散)和组织 用荧光法检测活细胞胞浆内的蛋白质、DNA和核糖体 显微镜。为了产生突然的渗透胁迫，我们使大肠杆菌细胞增加 外源盐浓度(质壁分离法)。或者，我们可以允许细胞逐渐 适应高盐环境下的生长。在适应的细胞中，类核 (染色体DNA)保持扩张，GFP的扩散仍然容易。在 质壁细胞，类核致密(收缩到小得多的体积)，以及 绿色荧光蛋白的扩散受到严重阻碍。蛋白质在体内扩散的能力 胞浆间隙可能决定细胞从渗透压中恢复的能力 冲击和恢复增长和分裂。 值得注意的是，关于如何将动力学和热力学结果从 稀释溶液以适应拥挤、复杂的细胞质环境。我们假设 一个双域模型(类核和细胞质外围)，其中空间 许多球状蛋白质的分布取决于类核的详细结构， 尤其是其对蛋白质的孔隙度 大小和装药量都不同。然后，平均轴向扩散系数就是加权平均值 随着时间的推移，在类核与外周之间。我们将测量空间 类核糖体和核糖体的分布及其扩散系数 活的大肠杆菌细胞质中的蛋白质，无论是在正常生长还是作为 渗透胁迫。这将极大地阐明大分子拥挤和 限制蛋白质扩散。一种新型的单细胞流动装置将 测量蛋白质扩散率和类核及细胞质大小的时间依赖关系 和在同一细胞中的形状，在质壁分离前后。基于Simple的计算 物理模型和实验测量的约束将提供更多 更好地理解核糖体与类核糖体的分离以及 球状蛋白在类核细胞和外周细胞质之间的分配 基于大小、电荷和DNA结合倾向。从长远来看，我们的方法可以 被扩展到时间依赖的药物对细胞质组织和 蛋白质在新的细节水平上的扩散。

This section has changed only slightly from the original proposal. The ubiquity of bacteria in nature attests to their tremendous adaptability. E. coli must routinely respond to external stress in the form of temperature change, nutrient deprivation, the presence of a drug, or a change in external solute concentration (osmotic stress). In this project we study protein transport (diffusion) and the organization of proteins, DNA, and ribosomes within the cytoplasm of live cells using fluorescence microscopy. To create a sudden osmotic stress, we subject E. coli cells to an increase in external salt concentration (plasmolysis). Alternatively, we can allow cells to gradually adapt to growth in high salt concentration. In the adapted cells, the nucleoid (chromosomal DNA) remains expanded, and diffusion of GFP remains facile. In the plasmolyzed cells, the nucleoid compacts (shrivels to a much smaller volume), and diffusion of GFP is severely hindered. The ability of proteins to diffuse through the plasmolyzed cytoplasmic space may determine the cell's ability to recover from osmotic shock and resume growth and division. Remarkably little is known about how to extend kinetics and thermodynamic results from dilute solutions to the crowded, complex environment of the cytoplasm. We hypothesize a two-domain model (nucleoids and cytoplasmic periphery) in which the spatial distribution of many globular proteins depends on the detailed structure of the nucleoid, especially on its porosity to proteins of different size and charge. The mean axial diffusion coefficient is then a weighted average over time spent within the nucleoid vs the periphery. We will measure the spatial distribution of nucleoids and ribosomes and the diffusion coefficient of a range of proteins in the cytoplasm of live E. coli both in normal growth and as a function of osmotic stress. This will greatly clarify the impact of macromolecular crowding and confinement on protein diffusion. A novel single-cell flow device will measure the time dependence of protein diffusivity and of nucleoid and cytoplasmic size and shape in the same cell, before and after plasmolysis. Calculations based on simple physical models and constrained by experimental measurements will provide a much better understanding of the segregation of ribosomes from the nucleoids and of the partitioning of globular proteins between the nucleoids and the peripheral cytoplasm based on size, charge, and DNA-binding propensity. In the longer term, our methods can be extended to the study of time-dependent drug effects on cytoplasmic organization and protein diffusion at a new level of detail.