Assembly of bacterial cytoplasmic protein complexes measured by LRET

描述（由申请人提供）：细菌细胞质含有与致病性和毒力相关的显著有序结构。由蛋白质MreB组成的肌动蛋白样细丝横跨细菌杆的长度。细胞器样细菌微区室（BMC），由几何蛋白质壳包围，隔离挥发性或有毒的反应中间体。许多革兰氏阴性病原体中的齿轮状小管有助于将毒素分泌到靶细胞中。关于这些结构在体内的组装和拆卸，只有有限的信息可用。发光共振能量转移（LRET）将用于测试肌动蛋白样丝组装和细菌微区室和微管结构和动力学的拟议模型。最近发现，大肠杆菌在发光铽离子与基因编码位点结合而对细胞无毒性的条件下吸收这些离子。拟议的实验将利用这一发现来研究活细胞中的分子结构。MreB和微区室壳蛋白EutL和EutM将用tetraCys序列标记，其随后将结合荧光素砷以产生LRET受体。铽将通过细胞质蛋白上的镧系元素结合标签（LBT）进行配位，所述细胞质蛋白将充当LRET供体。由于镧系元素的长激发态寿命，LRET对供体和受体分子的扩散速率敏感。当受体从有序结构释放到细胞质中时，应检测到LRET的大幅增加，而当受体从细胞质中移除到有序结构中时，应检测到LRET的大幅降低。这将提供在细胞分裂和代谢诱导期间可以遵循的结构组装和拆卸的光谱信号。还将在肠道沙门氏菌和霍乱弧菌中测量铽摄取。如果铽的吸收与E.大肠杆菌中进行了观察，还将对S.肠Pdu微区室和V. cholesterol VI型分泌系统小管。这些实验的结果将提供有关细菌肌动蛋白样细丝、微区室和小管如何在活细胞中组装的基本信息。这些信息将有助于未来靶向这些结构，以削弱肠杆菌的致病作用。

DESCRIPTION (provided by applicant): Bacterial cytoplasm contains remarkable ordered structures that are linked to pathogenicity and virulence. Actin-like filaments, made of the protei MreB, span the length of bacterial rods. Organelle-like bacterial microcompartments (BMCs), bounded by geometric protein shells, sequester volatile or toxic reaction intermediates. Cogwheel shaped tubules in many gram-negative pathogens help secrete toxins into targeted cells. Only limited information is available about the assembly and disassembly of these structures in vivo. Luminescence resonance energy transfer (LRET) will be used to test proposed models for actin-like filament assembly and bacterial microcompartment and tubule structure and dynamics. Recently it was discovered that Escherichia coli takes up luminescent terbium ions under conditions where these ions bind to genetically encoded sites without toxicity to the cells. The proposed experiments will exploit this finding to study molecular structures in live cells. MreB and the microcompartment shell proteins EutL and EutM will be tagged with tetraCys sequences, which will subsequently bind fluorescein arsenical to generate LRET acceptors. Terbium will be coordinated by a lanthanide-binding tag (LBT) on a cytoplasmic protein that will serve as the LRET donor. Because of the long excited state lifetimes of lanthanides, LRET is sensitive to the diffusion rates of donor and acceptor molecules. When the acceptors are released from ordered structures into the cytoplasm, a large increase in LRET should be detected, and when the acceptors are removed from the cytoplasm into ordered structures, a large decrease in LRET should be detected. This will provide spectroscopic signals of structure assembly and disassembly that can be followed during cell division and metabolic induction. Terbium uptake will also be measured in Salmonella enterica and Vibrio cholerae. If terbium uptake similar to E. coli is observed, studies will also be done on assembly of S. enterica Pdu microcompartments and V. cholerae type VI secretory system tubules. The results of these experiments will provide fundamental information about how bacterial actin-like filaments, microcompartments, and tubules are assembled in live cells. This information will aid future targeting of these structures to weaken the pathogenic effects of enterobacteria.