GENETIC COMPETENCE APPARATUS OF BACILLUS SUBTILLIS

枯草芽孢杆菌能被外源DNA转化 称为能力的生理状态。有能力的细胞能够结合 双链DNA，细胞表面的DNA碎片，并运输 穿过细胞被膜层的单链，伴随着释放 另一条链的酸溶产物。我们的长期目标是 在分子水平上理解这些过程。以前的工作有 确定了几个似乎是结合所必需的基因， 加工和/或运输转化的DNA。在大多数情况下，这些基因 已经被克隆、测序、转录作图，以及它们的调控 学习。除了一个例外，所有预测的基因产物都是 疏水性的，很可能是膜相关的。我们将使用组合的 用遗传、生化和免疫学方法分析其特性 其中的几种蛋白质，并确定它们的细胞位置。 Comg ORF1产物与农杆菌VIRB ORF11类似 Tumefaciens T1-质粒。后者的产物已被证明是一种ATPase。 我们将纯化Comg ORF1产品，确定它是否也是ATPase， 如果是这样的话，使用突变的方法来分析这种酶的作用 在几个与能力相关的职能部门中的活动。我们还将确定 这种蛋白质的细胞位置。类似的实验将分析 Comg ORF3在转化中的作用。本产品与 假单胞菌菌毛的一类。我们将研究在 能力特异性单链结合蛋白的转化，即 已经在文献中描述过了。我们将使用UV交联剂 一种分离能力特异性DNA结合蛋白或受体的方法 很复杂。最后，我们将利用电子显微镜来研究细胞 几种能力蛋白的位置和相互关系。

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.