GENETIC COMPETENCE APPARATUS OF BACILLUS SUBTILIS

枯草芽孢杆菌遗传感受态装置

• 批准号: 2182162
• 负责人: DAVID A DUBNAU
• 金额: $ 41.65万
• 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 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182162
• 关键词: Bacillus subtilis; DNA binding protein; G protein; adenosinetriphosphatase; bacterial genetics; biotransformation; cell membrane; cellular polarity; crosslink; electrophysiology; enzyme activity; genetic transduction; immunologic assay /test; intracellular transport; membrane proteins; mutant; nuclease; operon; phenotype; protein biosynthesis; protein degradation; protein structure function; protein transport; scanning electron microscopy

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 layers, with concomitant release of acid soluble products from the other strand. Our long term objective is to understand these processes at the molecular level. Previous work has identified a dozen gene products that appear to be necessary for the binding, processing and uptake of transforming DNA. The genes for these products have been cloned, sequenced and transcriptionally mapped, and their regulation studied. Most are predicted to be membrane proteins from their sequences. Several competence proteins have been shown to be membrane-localized using antibody reagents, and the nature of their membrane attachment has been characterized. The comG operon encodes seven proteins which appear to be required for the binding of DNA to the cell surface. The comE and comF operons encode at least two proteins which are required for the uptake but not the binding of DNA. The ComFI protein resembles known DNA translocases and helicases, and may utilize the energy of ATP hydrolysis to drive DNA uptake. The ComE3 protein is suggested to be a pore-forming protein. We will use combined genetic, biochemical, immunological, electrophysiological and high resolution electron microscopic approaches to explore the arrangements of these proteins on the cell surface, their interrelationships and their functional roles during transformation. We will use cross-linking to detect proteins that contact DNA during transformation. We will investigate the polarity of DNA uptake, the role of the Competence.specific nuclease activity, and we will seek additional competence gene products. Finally we will attempt to establish an in vitro DNA transport system.

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，在细胞表面上的DNA片段，和运输 单链穿过细胞包膜层，伴随着释放 另一股的酸溶性产物。长期不变的宗旨和追求 就是在分子水平上理解这些过程。以前的工作 已经确定了十几种基因产物，这些产物似乎是 转化DNA的结合、加工和摄取。这些基因 产品已被克隆，测序和转录图谱， 他们的规则研究。大多数被预测为膜蛋白， 他们的序列。几种感受态蛋白已被证明是 使用抗体试剂的膜定位，及其性质 已经表征了膜附着。comG操纵子编码7个 DNA与细胞结合所需的蛋白质 面comE和comF操纵子编码至少两种蛋白质， 这是摄取所需的，但不是DNA的结合。ComFI蛋白 类似于已知的DNA移位酶和解旋酶，并且可以利用 ATP水解的能量驱动DNA摄取。ComE3蛋白是 可能是一种造孔蛋白。我们将使用基因， 生物化学、免疫学、电生理学和高分辨率 电子显微镜的方法来探索这些安排 细胞表面的蛋白质，它们之间的相互关系， 在转型过程中发挥作用。我们将使用交叉连接， 检测在转化过程中与DNA接触的蛋白质。我们将 研究DNA摄取的极性， 特异性核酸酶活性，我们将寻求额外的 感受态基因产物最后，我们将尝试建立一个 体外DNA转运系统