GENETIC COMPETENCE APPARATUS OF BACILLUS SUBTILLIS

枯草芽孢杆菌遗传感受态装置

• 批准号: 3302783
• 负责人: DAVID A DUBNAU
• 金额: $ 36.93万
• 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 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302783
• 关键词: Agrobacterium tumefaciens; Bacillus subtilis; DNA binding protein; Pseudomonas; adenosinetriphosphatase; bacterial genetics; binding proteins; cell transformation; crosslink; enzyme biosynthesis; enzyme mechanism; enzyme substrate complex; gene mutation; membrane proteins; pilin; plasmids; protein biosynthesis; protein kinase; protein purification; protein transport; site directed mutagenesis; ultraviolet radiation

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 of the other strand. Our long term objective is to understand these processes on the molecular level. Previous work has identified several genes that appear to be necessary for the binding, processing and/or transport of transforming DNA. In most cases, these genes have been cloned, sequenced, transcriptionally mapped, and their regulation studied. With one exception, all of the predicted gene products are hydrophobic, and likely to be membrane associated. We will use a combined genetic, biochemical and immunological approach to analyze the properties of several of these proteins, and to determine their cellular locations. The comG ORF1 product is similar to that of virB ORF11 of the Agrobacterium tumefaciens T1-plasmids. The latter product has been shown to be an ATPase. We will purify the comG ORF1 product determine whether it too is an ATPase, and if so, use a mutagenic approach to analyzing the role of this enzymatic activity in several competence-related functions. We will also determine the cellular location of this protein. Similar experiments will analyze the role of comG ORF3 in transformation. This product is related to the Pseudomonas class of bacterial pilins. We will study the possible role in transformation of a competence-specific single strand binding protein, that has been described in the literature. We will use a UV cross-linking approach to isolate a competence-specific DNA binding protein or receptor complex. Finally, we will utilize electron microscopy to study the cellular locations and interrelationships of several competence proteins.

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 另一条链的酸溶性产物。我们的长远目标是 在分子水平上理解这些过程。先前的工作已经 确定了几个似乎是结合所必需的基因， 转化DNA的加工和/或运输。在大多数情况下，这些基因 已经被克隆，测序，转录图谱，以及它们的调控 研究了除了一个例外，所有预测的基因产物都是 疏水的，并且可能是膜相关的。我们将使用一个组合 遗传学、生物化学和免疫学方法来分析特性 这些蛋白质中的几种，并确定它们的细胞位置。 comG ORF 1产物与农杆菌属的virB ORF 11产物相似 tumefaciens T1质粒。后一种产物已被证明是ATP酶。 我们将纯化comG ORF 1产物，以确定它是否也是ATP酶， 如果是的话，用诱变方法来分析这种酶的作用， 在几个与能力有关的职能中开展活动。我们还将确定 这种蛋白质的细胞定位。类似的实验将分析 comG ORF 3在转化中的作用。该产品与 假单胞菌类细菌菌毛蛋白。我们将研究在以下方面的可能作用： 转化能力特异性单链结合蛋白， 已经在文献中描述过。我们将使用紫外线交联 分离能力特异性DNA结合蛋白或受体的方法 复杂.最后，我们将利用电子显微镜研究细胞 几种感受态蛋白的位置和相互关系。