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.
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