Genetic Competence Apparatus of Bacillus Subtilis

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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.