Structural and Dynamical Response of Escherichia coli to Osmotic Stress

本节与原提案相比仅略有改动。 细菌在自然界中的普遍存在证明了它们巨大的适应性。e.大肠杆菌必须 通常会对外界压力作出反应，如温度变化、营养物质 剥夺，药物的存在，或外部溶质浓度（渗透压）的变化 应力）。在这个项目中，我们研究蛋白质运输（扩散）和组织的 活细胞胞质内的蛋白质、DNA和核糖体， 显微镜为了产生突然的渗透压，我们使E。大肠杆菌细胞增加， 外盐浓度（plasma）。或者，我们可以让细胞逐渐 适应高盐浓度生长。在适应的细胞中， （染色体DNA）保持扩增，并且GFP的扩散保持容易。在 质体化的细胞，类核压缩（萎缩到更小的体积）， GFP的扩散受到严重阻碍。蛋白质扩散通过 浆化的细胞质空间可能决定细胞从渗透压恢复的能力。 冲击并恢复增长和分裂。 对于如何扩展动力学和热力学结果，人们知之甚少。 稀释的溶液到拥挤的，复杂的细胞质环境。我们假设 两域模型（类核和细胞质外围），其中空间 许多球状蛋白的分布取决于类核的详细结构， 特别是它对蛋白质的多孔性， 不同的大小和收费。平均轴向扩散系数是加权平均值 随着在类核内相对于外围花费的时间的变化。我们将测量空间 类核和核糖体的分布和扩散系数的范围内， 活E.大肠杆菌在正常生长和作为一个功能 渗透胁迫这将大大澄清大分子拥挤的影响， 限制蛋白质扩散。一种新颖的单细胞流动装置将 测量蛋白质扩散率以及类核和细胞质大小的时间依赖性 在同一个细胞中，在plastomerase之前和之后。计算基于简单 物理模型和实验测量的约束将提供一个很大的 更好地理解核糖体从类核中的分离， 球状蛋白在类核和周围细胞质之间的分配 基于大小电荷和DNA结合倾向从长远来看，我们的方法可以 扩展到研究药物对细胞质组织的时间依赖性影响， 蛋白质扩散在一个新的细节水平。

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.