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性DNA转化 生理状态被称为能力。胜任的单元能够结合 双链DNA，碎片在细胞表面上的DNA，然后转运 跨细胞包膜层的一条链，并释放 另一链的酸溶剂产物。我们的长期目标是 在分子水平上了解这些过程。以前的工作有 确定了几个似乎是结合所必需的基因， 转化DNA的处理和/或运输。在大多数情况下，这些基因 已被克隆，测序，转录映射，其调节 研究。除一个例外，所有预测的基因产物都是 疏水，可能与膜相关。我们将共同使用 遗传，生化和免疫学方法分析特性 其中几种蛋白质，并确定其细胞位置。 COMG ORF1产品与农业的VirB ORF11相似 Tumefaciens T1-质粒。后一种产品已显示为ATPase。 我们将净化COMG ORF1产品确定它是否也是ATPase， 如果是这样，请使用诱变方法分析这种酶促的作用 在几个与能力有关的功能中的活动。我们还将确定 该蛋白质的细胞位置。类似的实验将分析 COMG ORF3在转换中的作用。该产品与 细菌PILIN的假单胞菌类。我们将研究可能的作用 能力特异性单链结合蛋白的转化， 在文献中已经描述了。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 复杂的。最后，我们将利用电子显微镜研究细胞 几种能力蛋白的位置和相互关系。