Elucidating the spatiotemporal regulation of septal peptidoglycan synthases in E.coli

阐明大肠杆菌中隔膜肽聚糖合酶的时空调节

• 批准号: 10680050
• 负责人: Amilcar Josue Perez
• 金额: $ 7.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680050
• 关键词: Back; Bacterial Antibiotic Resistance; Cell Shape; Cell Wall; Cell division; Cells; Complex; Cytolysis; Cytoprotection; Cytoskeletal Proteins; Enzyme Kinetics; Enzymes; Equilibrium; Escherichia coli; Exhibits; Frequencies; Future; Genetic; Geometry; Glucosyltransferase; Goals; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Hydrolase; Hydrolysis; Immobilization; Individual; Kinetics; Knowledge; Light; Lighting; Lipids; Measurement; Measures; Microscopy; Molecular; Morphogenesis; Morphology; Motion; Movement; Multienzyme Complexes; Osmosis; Outcome; Pathway interactions; Peptidoglycan; Peptidyltransferase; Polymers; Population; Process; Protein Biosynthesis; Protein Dynamics; Proteins; Regulation; Regulatory Pathway; Role; Scanning Electron Microscopy; Scheme; Shapes; Signal Transduction; Stress; Structure; Time; Tubulin; Variant; Work; analysis pipeline; constriction; crosslink; daughter cell; driving force; dynamic system; enzyme activity; genetic variant; glycosyltransferase; imaging study; insight; molecular imaging; novel; protein phosphatase inhibitor-2; protein protein interaction; recruit; single molecule; spatiotemporal; tool; treadmill; z-ring

PROJECT SUMMARY In E. coli, cell division occurs at the midcell via initial assembly of the division apparatus into a septal ring and subsequent septal ring closure. The septal ring contains essential proteins including FtsZ (tubulin homolog an GTPase), FtsW (glycosyltransferase), FtsI (transpeptidase), and regulators including FtsN and the FtsBLQ complex. Together FtsW and FtsI form a septal peptidoglycan (sPG) synthase complex (FtsWI) that is essential for new septum synthesis. Recently, single molecules imaging studies in live E. coli cells revealed three different states of FtsWI along the septum: two processive moving states and one immobile state. The slow processive movement of FtsWI is driven by their own sPG synthesis activities by which PG-substrate (Lipid II) is continuously polymerized and crosslinked to the existing septal cell wall (termed on sPG track). The fast processive movement of FtsWI is driven by FtsZ’s treadmilling dynamics but not active in sPG synthesis (termed on the Z-track). Additionally, some FtsWI molecules can display an “immobile” state whereby they remain stationary at septum, but can transition into the fast- and slow-moving populations. These different mobility states of FtsWI complexes signal different states of their activities, and hence providing a new way to investigate the activity regulation pathway of FtsWI at the septum and the corresponding spatiotemporal coordination in septum synthesis. The broad goal of this work is to characterize the spatiotemporal regulation of FtsWI in live E. coli cells and investigate how such regulation impact septum cell wall synthesis and pole morphogenesis. In Aim 1, I will determine the kinetic pathway of FtsWI activation in live cells by developing a single-molecule imaging and analysis pipeline and measure transition frequencies between different states and the corresponding state lifetimes of FtsWI. Using this pipeline, I will investigate how lipid II levels or protein modulators of FtsWI differentially feed into the regulatory pathway. In Aim 2 I will investigate how the relative distributions of FtsWI on the sPG and Z-tracks are related to division progression and cell pole morphogenesis. Using a combination of single molecule tracking, Structured Illumination Microscopy (SIM), Scanning Electron Microscopy, and genetic and growth conditions known to unbalance the ratio of FtsWI on the two tracks, I will determine to what extent the two tracks are required to maintain balanced rates of septum closure and septum symmetry that give rise to cell pole shape. Elucidating the kinetic pathway for FtsWI regulation will reveal novel insights regarding how molecular inputs feed into this dynamic system.

项目摘要 在大肠在大肠杆菌中，细胞分裂发生在中细胞，通过分裂器最初组装成隔环 以及随后的间隔环闭合。隔环含有必需的蛋白质，包括FtsZ（微管蛋白同源物 GT3）、FtsW（糖基转移酶）、FtsI（转肽酶）和包括FtsN和FtsBLQ的调节剂 复杂. FtsW和FtsI共同形成间隔肽聚糖（sPG）合酶复合物（FtsWI）， 用于新隔膜合成。近年来，单分子成像技术在活体大肠杆菌中的应用越来越广泛。大肠杆菌细胞揭示了三种不同的 FtsWI沿隔沿着的运动状态：两种进行性运动状态和一种静止状态。缓慢进行 FtsWI的运动是由它们自身的sPG合成活动驱动的，PG-底物（脂质II）通过该活动被连续地 聚合并交联到现有的隔膜细胞壁（在sPG轨道上称为）。快速进行运动 的FtsWI是由FtsZ的研磨动力学驱动的，但在sPG合成中不活跃（在Z轨道上被称为）。 此外，一些FtsWI分子可以显示“不动”状态，由此它们在隔膜处保持静止， 但可以过渡到快速和缓慢移动的种群。FtsWI复合物的这些不同迁移率状态 信号的不同活动状态，从而提供了一个新的途径来研究活动的调节 FtsWI在隔区的信号传导途径及其在隔区合成中的时空协调性。 这项工作的主要目标是表征FtsWI在活大肠杆菌中的时空调节。杆菌细胞 并研究这种调节如何影响隔膜细胞壁合成和极形态发生。在目标1中，我将 通过开发单分子成像来确定活细胞中FtsWI激活的动力学途径， 分析流水线并测量不同状态与对应状态之间的转换频率 FtsWI的寿命。使用这个管道，我将研究FtsWI的脂质II水平或蛋白质调节剂如何 差异化地进入调节途径。在目标2中，我将研究FtsWI的相对分布如何 sPG和Z-轨道上的信号与分裂进程和细胞极形态发生有关。使用组合 单分子跟踪、结构照明显微镜（SIM）、扫描电子显微镜和 遗传和生长条件已知不平衡的比例FtsWI的两个轨道上，我将确定什么 在一定程度上，需要两个轨道来保持隔膜闭合的平衡速率和隔膜对称性， 上升到细胞极形状。阐明FtsWI调控的动力学途径将揭示关于FtsWI调控的新见解。 分子输入是如何进入这个动态系统的