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

项目总结/摘要 多种蛋白质的空间和时间协调对于调节细胞内的复杂过程至关重要。 细菌细胞，包括用于细胞伸长和细胞分裂的肽聚糖合成，形态发生，细胞 分化、外部结构的生物发生、对表面的粘附和生物膜形成以及老化。的 本研究的主要目的是确定控制时空组织的机制， 以及这种组织如何转化为有益于健康的表型。该项目有三 主要部件：1.肽聚糖合成时空动力学调控机制的研究 在不同的区域来驱动细胞伸长、分裂和形态发生。标记位点的荧光探针 将优化肽聚糖合成，使实验增加空间和时间 分辨率间隔肽聚糖合成模式将通过连续标记肽聚糖进行研究 不同颜色的探测器，其空间模式将提供PG区域的时间顺序说明 在每个脉冲标记期间合成。改变FtsZ螺纹铣削的效果和PBP 2b的运动 将测试间隔PG合成蛋白对肽聚糖合成速度的影响。SpmX的功能 morphogen，它指定肽聚糖合成的小区域，以产生薄的圆柱形延伸， 细胞被称为茎，将通过确定其结构，其定位机制， 识别相互作用的蛋白质。2.蛋白质定位和细胞凋亡机制的研究 不对称性调节细胞周期、细胞分化和衰老。一种新的组氨酸调控机制 通过极性支架蛋白PodJ去磷酸化的激酶将使用生物化学方法进行研究。 方法和诱变以确定其作用机制。PodJ的磁极定位机制 并将研究其降解以释放组氨酸激酶的抑制。细胞不对称性在 老化将通过研究其对损伤隔离的影响来确定。3.研究了其作用机制， 细菌对表面的粘附性和强粘合剂的生物化学性质。的作用 新月柄杆菌鞭毛和皮利在表面感受和介导从可逆性 到粘合力的永久阶段，最终在粘合剂固着剂的合成，将进行研究， 它们在各种突变体的粘附过程中的定量跟踪。生物物理学基础 将通过原子力显微镜动态力来研究固着粘合剂的令人印象深刻的强度 通过电子束蚀刻或离子束的组合对其结构进行光谱和高分辨率分析 研磨、AFM成像和纳米压痕。从这些研究中获得的见解可以用于设计 抑制生长、防止关键形态学变化或改变重要蛋白定位途径的策略 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.