GENETIC COMPETENCE APPARATUS OF BACILLUS SUBTILIS

枯草芽孢杆菌遗传感受态装置

• 批准号: 6698351
• 负责人: DAVID A DUBNAU
• 金额: $ 17.19万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-01 至 2003-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6698351
• 关键词: Bacillus subtilis; DNA binding protein; DNA replication; Xenopus oocyte; bacterial genetics; bacterial proteins; chromosome movement; crosslink; gene mutation; immunoprecipitation; intracellular transport; operon; protein protein interaction; surface plasmon resonance; yeasts

DESCRIPTION (adapted from the investigator's abstract): Bacillus subtilis can be transformed by exogenous DNA when in a physiological state known as competence. Competent cells are able to bind double-stranded DNA, fragment the DNA on the cell surface, and transport a single strand across the cell envelope. The long-term objective of this project is to understand these processes on the molecular level. A dozen genes required for transformation, and their protein products, have been characterized. The interactions among these proteins and their roles in DNA binding and transport will be investigated by genetic and biochemical methods. These will include mutagenesis of individual proteins followed by the search for allele- specific suppression, the analysis of protein/protein interactions using surface plasmon resonance, cross-linking and immunoprecipitation, and determination of the accessibility of a known competence DNA binding protein to exogenous molecular probes of varying sizes in mutants lacking other competence proteins. The fate of transforming DNA will be characterized in mutants lacking known competence proteins, and the proteins contacted by transforming DNA during binding and uptake will be characterized using a novel DNA-protein cross-linking approach. An in vitro DNA transport system will be developed using membrane vesicles. Additional genes required for transformation will also be isolated and characterized. The possibility that the type 4 pilin-like ComG proteins interact with and remodel the cell wall will also be investigated, as will the effect of the ComGA protein on replication and segregation of chromosomes in competent cells.

描述（改编自研究者的摘要）： 枯草芽孢杆菌可以被外源DNA转化 生理状态称为能力。感受态细胞能够 结合双链 DNA，使细胞表面的 DNA 片段化，并且 运输单链穿过细胞膜。长期来看 该项目的目标是了解这些过程 分子水平。转化所需的十几个基因及其 蛋白质产品，已被表征。这些之间的相互作用 蛋白质及其在 DNA 结合和运输中的作用将 通过遗传和生化方法进行研究。这些将包括 单个蛋白质的诱变，然后寻找等位基因 特异性抑制，使用分析蛋白质/蛋白质相互作用 表面等离子共振、交联和免疫沉淀，以及 确定已知 DNA 结合能力的可及性 突变体中不同大小的蛋白质与外源分子探针 缺乏其他能力蛋白。改造DNA的命运将是 以缺乏已知能力蛋白的突变体为特征，并且 在结合和摄取过程中通过转化 DNA 接触的蛋白质将 使用新型 DNA-蛋白质交联方法进行表征。一个 体外DNA转运系统将使用膜囊泡进行开发。 转化所需的其他基因也将被分离并 特点。 4 型菌毛蛋白样 ComG 蛋白的可能性 还将研究与细胞壁的相互作用和重塑，因为 ComGA 蛋白对复制和分离的影响 感受态细胞中的染色体。