BACTERIAL CHEMOTAXIS PROBED BY PHOTORELEASED LIGANDS

通过光释放配体探测细菌趋化性

• 批准号: 6053297
• 负责人: SHAHID M KHAN
• 金额: $ 4.62万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6053297
• 关键词: Escherichia coli; aminoacid; bacterial toxins; cell cell interaction; cell migration; chemoattractants; chemotaxis; cilium /flagellum motility; computer assisted diagnosis; computer program /software; electroporation; method development; phosphorylation; photolysis; sensory signal detection; video recording system

This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION: In a revision of an application, Dr. Khan proposes, as previously, to investigate sensory excitation and signal amplification in the chemotactic system of Escherichia coli using a rapid assay for measurement of chemotactic excitation that he has developed and that involves flash photo release of caged compounds and computer-assisted video analysis of cellular behavior. The overall goal is to understand signal amplification in terms of the intracellular phosphorylation cascade mediating signalling between the chemoreceptors and the flagellar motor. Photorelease of caged attractants (serine and aspartate) or repellents (protons and leucine) will be used to apply stimuli to free swimming cells at defined times and thus allow measurement of the speed and amplitude of the excitation response. Initial studies using caged serine have been recently published. This work will be extended to investigate response to small-step changes in stimulant concentration at different initial concentrations, and to investigate responses in cells with altered initial swim/tumble balances as the result of metabolic and mutational perturbations. Dr. Khan also proposes to continue work on the development of a computer program for automated analysis of a field of tethered cells. This would allow his proposed analyses of responses of swimming cells, representing a summation of action of the multiple flagella on each cell, to be compared to response of tethered cells, representing the action of a single flagellum. A second aim is to relate the kinetics of phosphotransfer in vitro to the kinetics of excitation in intact cells by examining excitation in mutants defective in interaction between CheY and the flagellar motor, in strains containing increased amounts of CheY or CheY plus CheZ or CheB, and in mutants defective in CheZ or in phosphotransfer between CheA and CheY. A third aim is to characterize the affects of the adaptation components on excitation by examining excitation in mutants altered in the CheB methylesterase or in the methyl-accepting site of a chemoreceptor. The fourth aim is to develop and characterize electroporation techniques that will allow dependable and informative experiments using caged compounds introduced into cells.

这是香农奖，为研究提供部分支持 与指定研究所的资金范围相差的项目，但 处于卓越的范围。香农奖旨在提供 支持测试方法的可行性；开发进一步的测试 并完善研究技术；对可用的次要分析 数据集；或执行可以证明PI的离散项目 研究能力或增加本来有功的重量 应用。下面的摘要取自原始文档 由首席调查员提交。 描述：在对应用程序的修订中，汗博士建议 以前，研究感觉激发和信号扩增 在大肠杆菌的趋化系统中，使用快速测定 他开发的趋化激发的测量 涉及笼子化合物和计算机辅助的闪光灯照片 细胞行为的视频分析。总体目标是了解 信号放大，以细胞内磷酸化 化学感受器和 鞭毛马达。笼中吸引剂的光松饼（丝氨酸和 天冬氨酸）或驱虫剂（质子和亮氨酸）将用于申请 刺激在定义的时间释放游泳单元，从而允许 测量激发响应的速度和振幅。 使用笼丝氨酸的初步研究最近已发表。这 将扩展工作以调查对小步变化的反应 在不同初始浓度下的刺激浓度，至 调查改变初始游泳/翻滚平衡的细胞中的反应 由于代谢和突变扰动的结果。可汗博士也是如此 建议继续开发用于开发计算机程序 自动分析束缚细胞领域。这将允许他 提出的游泳细胞反应的分析，代表 多个鞭毛在每个单元上的作用求和为 与束缚细胞的响应相比，代表A的作用 单个鞭毛。第二个目的是关联 在完整细胞中激发动力学的体外磷酸转移 通过检查Chey之间相互作用有缺陷的突变体的激发 和鞭毛电动机，含有增加chey量的应变 或Chey Plus Chez或Cheb，以及在Chez或在Chez中有缺陷的突变体 Chea和Chey之间的磷酸转移。第三个目标是表征 通过检查适应成分对激发的影响 在Cheb甲基酯酶中改变的突变体的激发或 化学感受器的甲基受感受部位。第四个目标是发展 并表征将允许可靠的电穿孔技术 并使用引入细胞中引入的笼化合物的信息实验。