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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要在大肠杆菌中，细胞分裂通过分裂装置初始组装成隔膜环而发生在细胞中部以及随后的隔膜环闭合隔膜环含有必需蛋白质，包括 FtsZ（微管蛋白同源物）。 GTPase）、FtsW（糖基转移酶）、FtsI（转肽酶）以及包括 FtsN 和 FtsBLQ 在内的调节因子 FtsW 和 FtsI 一起形成重要的隔膜肽聚糖 (sPG) 合酶复合物 (FtsWI)。最近，活大肠杆菌细胞的单分子成像研究揭示了三种不同的隔膜合成方法。 FtsWI 沿隔膜的状态：两种进行性移动状态和一种不动状态。 FtsWI 的运动是由其自身的 sPG 合成活动驱动的，通过这些活动，PG 底物（脂质 II）不断聚合并交联到现有的隔膜细胞壁（称为 sPG 轨道）。 FtsWI 的 FtsWI 由 FtsZ 的跑步动态驱动，但在 sPG 合成中不活跃（在 Z 轨道上称为）。此外，一些 FtsWI 分子可以表现出“不动”状态，即它们在隔膜上保持静止，但可以转变为 FtsWI 复合体的快速和缓慢移动群体。发出不同活动状态的信号，从而提供一种研究活动调节的新方法隔膜处的 FtsWI 通路以及隔膜合成中相应的时空协调。这项工作的总体目标是表征活大肠杆菌细胞中 FtsWI 的时空调节并研究这种调节如何影响隔膜细胞壁的合成和极的形态发生。通过开发单分子成像技术确定活细胞中 FtsWI 激活的动力学途径分析管道并测量不同状态和相应状态之间的转换频率 FtsWI 的生命周期，我将研究 FtsWI 的脂质 II 水平或蛋白质调节剂如何变化。在目标 2 中，我将研究 FtsWI 的相对分布如何。 sPG 和 Z 轨道上的变化与分裂进程和细胞极形态发生有关。单分子追踪、结构照明显微镜 (SIM)、扫描电子显微镜和已知遗传和生长条件会使两条轨道上的 FtsWI 比例失衡，我将确定什么两个轨道需要保持平衡的隔膜闭合率和隔膜对称性，从而达到一定程度阐明 FtsWI 调节的动力学途径将揭示关于细胞极形状的新见解。分子输入如何进入这个动态系统。