Bacterial Subcellular Organization and its Impact on Growth, Development, Aging, and Surface Adhesion

项目摘要/摘要 多个蛋白质的空间和时间协调对于调控复杂的过程是至关重要的 细菌细胞，包括用于细胞伸长和细胞分裂、形态发生、细胞生长的肽聚糖合成 分化，外部结构的生物发生，表面黏附和生物膜形成，以及老化。这个 这项研究的主要目的是确定控制组织的时空机制 细菌细胞以及这种组织如何转化为有益于健康的表型。该项目有三个 主要研究内容：1.肽多糖合成的时空动力学控制机制研究 在不同区域驱动细胞伸长、分裂和形态发生。标记这些地点的荧光探针 将对肽聚糖的合成进行优化，以便能够在空间和时间上增加实验 决议。隔膜肽聚糖的合成模式将通过用肽聚糖连续标记来研究 不同颜色的探测器，其空间模式将提供PG区域的时间顺序 在每个脉冲标记期间进行合成。不同FtsZ螺纹磨对PBP2b运动的影响 对隔膜PG合成蛋白中肽聚糖的合成速度进行了测试。SpmX的功能 Morogen，它指定了合成肽聚糖的小区域，以生成细长的圆柱形延伸部分 将通过确定细胞被膜的结构、定位机制和 鉴定相互作用的蛋白质。2.蛋白质定位和细胞定位的机制研究 不对称调节细胞周期、细胞分化和衰老。组氨酸调节的一种新机制 将使用生化方法研究由极性支架蛋白PodJ去磷酸化所致的激酶 方法和诱变，以确定其作用机制。PodJ定位于极点的机制 并将研究其降解以释放对组氨酸激酶的抑制作用。细胞不对称性在细胞生长发育中的作用 老化将通过研究其对损伤偏析的影响来确定。3.对黄褐斑形成机制的研究 细菌对表面的粘附性和强粘性的生物化学特性。美国政府的角色 新月形杆菌鞭毛和菌毛的表面感觉及其在介导可逆性转变中的作用 到永久粘合阶段，最终合成粘合剂夹持器，将通过以下方法进行研究 它们在各种突变体的黏附过程中的定量示踪。其生物物理基础 将用原子力显微镜动态力来研究固定胶粘剂的惊人强度 电子束刻蚀或离子束联合应用的光谱和高分辨率结构分析 球磨、原子力显微镜成像和纳米压痕。从这些研究中获得的见解可以用来设计 抑制生长、防止关键的形态变化或改变重要的蛋白质定位途径的策略 病原体，从而提高我们控制它们的能力。

Project Summary/Abstract The spatial and temporal coordination of multiple proteins is critical for the regulation of complex processes in bacterial cells, including peptidoglycan synthesis for cell elongation and cell division, morphogenesis, cell differentiation, biogenesis of external structures, adhesion to surfaces and biofilm formation, and aging. The main goal of this study is to determine the mechanisms that control the spatio-temporal organization of bacterial cells and how this organization is translated into phenotypes that benefit fitness. The project has three major parts: 1. A study the mechanisms that control the spatio-temporal dynamics of peptidoglycan synthesis in different zones to drive cell elongation, division, and morphogenesis. Fluorescent probes that label the sites of peptidoglycan synthesis will be optimized to enable experiments with increased spatial and temporal resolution. Septal peptidoglycan synthesis patterns will be studied by successive labeling with peptidoglycan probes of different colors, whose spatial pattern will provide a chronological account of the areas of PG synthesis during each pulse labeling. The effect of varying FtsZ threadmilling and the movement of the PBP2b septal PG synthesis protein on the velocity of peptidoglycan synthesis will be tested. The function of the SpmX morphogen, which specifies small zones of peptidoglycan synthesis to generate thin cylindrical extensions of the cell envelope called stalks, will be studied by determining its structure, its localization mechanisms, and by identifying interacting proteins. 2. A study of the mechanisms by which protein localization and cellular asymmetry regulate the cell cycle, cell differentiation, and aging. A novel mechanism of regulation of histidine kinases by dephosphorylation by the polar scaffold protein PodJ will be investigated using biochemical approaches and mutagenesis to determine its the mechanism. The mechanism of PodJ localization to the pole and its degradation to release inhibition of the histidine kinase will be studied. The role of cellular asymmetry in aging will be determined by studying its impact on damage segregation. 3. A study of the mechanisms of bacterial adhesion to surfaces and the biochemical properties of a strong adhesive. The role of the Caulobacter crescentus flagellum and pili in surface sensing and in mediating the transition from the reversible to the permanent phase of adhesion, culminating in the synthesis of an adhesive holdfast, will be studied by their quantitative tracking during the adhesion process of various mutants. The biophysical basis for the impressive strength of the holdfast adhesive will be studied by atomic force microscopy dynamic force spectroscopy and high resolution analysis of its structure by a combination of E-beam etching or ion beam milling, AFM imaging, and nanoindentation. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them.