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

细菌亚细胞组织及其对生长、发育、衰老和表面粘附的影响

• 批准号: 9276966
• 负责人: YVES V BRUN
• 金额: $ 76.21万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276966
• 关键词: Adhesions; Adhesives; Aging; Area; Atomic Force Microscopy; Bacterial Adhesion; Biochemical; Biogenesis; Biophysics; Caulobacter crescentus; Cell Cycle; Cell Differentiation process; Cell Shape; Cell division; Cells; Chronology; Color; Complex; Development; Fluorescent Probes; Goals; Growth; Growth and Development function; Image; Ions; Label; Mediating; Microbial Biofilms; Morphogenesis; Morphology; Movement; Mutagenesis; Pathway interactions; Pattern; Peptidoglycan; Phase; Phenotype; Physiologic pulse; Pilum; Process; Property; Protein Biosynthesis; Protein Dephosphorylation; Proteins; Regulation; Resolution; Role; Scaffolding Protein; Site; Specific qualifier value; Spectrum Analysis; Structure; Surface; Testing; Thinness; Translating; adhesion process; cell envelope; cell growth; design; experimental study; fitness; improved; insight; morphogens; mutant; nanoindentation; novel; pathogen; prevent; protein-histidine kinase; segregation; spatiotemporal; temporal measurement

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.

项目概要/摘要 多种蛋白质的空间和时间协调对于复杂过程的调节至关重要 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、表面粘附和生物膜形成以及老化。这 本研究的主要目标是确定控制时空组织的机制 细菌细胞以及该组织如何转化为有益于健康的表型。该项目共有三 主要内容： 1.研究肽聚糖合成时空动态的控制机制 在不同区域驱动细胞伸长、分裂和形态发生。标记位点的荧光探针 肽聚糖合成的过程将得到优化，使实验能够增加空间和时间 解决。将通过肽聚糖连续标记来研究间隔肽聚糖合成模式 不同颜色的探针，其空间图案将提供 PG 区域的时间顺序说明 每个脉冲标记期间的合成。不同 FtsZ 螺纹铣削和 PBP2b 运动的效果 将测试隔膜PG合成蛋白对肽聚糖合成速度的影响。 SpmX 的功能 形态发生素，它指定肽聚糖合成的小区域以产生薄的圆柱形延伸 称为茎的细胞包膜将通过确定其结构、定位机制以及通过 识别相互作用的蛋白质。 2. 蛋白质定位和细胞作用机制的研究 不对称性调节细胞周期、细胞分化和衰老。组氨酸调节的新机制 将使用生化方法研究通过极性支架蛋白 PodJ 去磷酸化的激酶 方法和诱变以确定其机制。 PodJ 极点定位机制 并将研究其降解以释放组氨酸激酶的抑制作用。细胞不对称的作用 老化将通过研究其对损伤隔离的影响来确定。 3. 机制研究 细菌对表面的粘附力和强粘合剂的生化特性。的作用 新月柄杆菌鞭毛和菌毛在表面传感和介导可逆转变中的作用 到粘合的永久阶段，最终合成粘合剂固定器，将通过以下方式进行研究 它们在各种突变体的粘附过程中的定量跟踪。的生物物理学基础 将通过原子力显微镜动态力研究固着粘合剂的令人印象深刻的强度 通过结合电子束蚀刻或离子束对其结构进行光谱和高分辨率分析 铣削、AFM 成像和纳米压痕。从这些研究中获得的见解可用于设计 抑制生长、防止关键形态变化或改变重要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。