Genetic Competence Apparatus of Bacillus Subtilis

描述(申请人提供)：细菌中的自然转化是指吸收和整合外源DNA，枯草芽孢杆菌可以被外源DNA转化时，在一种被称为感受态的生理状态下，合格细胞能够结合双链DNA，在细胞表面断裂DNA，并将单链运输穿过细胞膜，我们的长期目标是在分子水平上了解这些过程。该建议探讨了已知参与DNA转化加工和运输(内化)结合的蛋白质的具体作用，其中几种蛋白质(七种Comg基因产物、Comc和comeA)是将DNA与细胞表面结合所必需的，而其他蛋白质(comEC、comFA和comea)则是跨越细胞膜运输DNA所必需的。这项拟议的工作将探索这些蛋白质之间的相互作用，以及硫醇-二硫键氧化还原酶BDBD和BDBC在这些蛋白质的折叠和DNA摄取装置组装中的作用。一种新发现的高阶ComGC复合体的形成和性质将被阐明，并将研究它在DNA结合中的作用。可能的膜通道蛋白ComEC的结构和转运体ATPase ComFA的体外生化活性将进一步鉴定。将测定这些不同蛋白质在细胞中的数量，研究几种新鉴定的转化蛋白，最后尝试开发一种基于囊泡的体外DNA运输系统。

DESCRIPTION (provided by applicant): Natural transformation in bacteria refers to the uptake and integration of exogenous DNA, 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 membrane, Our long-term objective is to understand these processes on the molecular level. This proposal explores the specific roles of proteins known to participate in the binding on processing and transport (internalization) of transforming DNA, Several of these proteins (the seven comG gene products, ComC and ComEA) are needed for the binding of DNA to the cell surface while others (ComEC, ComFA and ComEA) are required for transport of DNA across the cell membrane. The proposed work will explore interactions among these proteins and the role of the thiol-disulfide oxidoreductases BdbD and BdbC in the folding of these proteins and in the assembly of the DNA uptake apparatus. The formation and properties of a newly identified higher order ComGC complex will be elucidated and its role in DNA binding will be examined. The structure of the putative membrane channel protein ComEC and the in vitro biochemical activities of the transporter ATPase ComFA will be characterized further. The amounts of these various proteins in the cell will be determined, Several newly identified transformation proteins will be studied, Finally an attempt will be made to develop a vesicle-based in vitro DNA transport system.