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（改编自研究者摘要）： 枯草芽孢杆菌可以通过外源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， 将单链穿过细胞膜。长期 本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， 蛋白质产品，已被表征。它们之间的相互作用 蛋白质及其在DNA结合和运输中的作用将在 通过遗传学和生物化学方法进行研究。这些将包括 对单个蛋白质进行诱变，然后寻找等位基因- 特异性抑制，蛋白质/蛋白质相互作用的分析，使用 表面等离子体共振、交联和免疫沉淀，以及 确定已知感受态DNA结合的可及性 突变体中不同大小的外源分子探针 缺乏其他感受态蛋白。改造DNA的命运将是 特征在于缺乏已知感受态蛋白的突变体， 在结合和摄取过程中与转化DNA接触的蛋白质将 使用新的DNA-蛋白质交联方法表征。一个 将利用膜囊泡开发体外DNA转运系统。 还将分离转化所需的其它基因， 表征了4型菌毛蛋白样ComG蛋白 与细胞壁的相互作用和重塑也将被研究， ComGA蛋白对复制和分离的影响 感受态细胞中的染色体。