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 from the other strand. Our long term objective is to understand these processes at the molecular level. Previous work has identified a dozen gene products that appear to be necessary for the binding, processing and uptake of transforming DNA. The genes for these products have been cloned, sequenced and transcriptionally mapped, and their regulation studied. Most are predicted to be membrane proteins from their sequences. Several competence proteins have been shown to be membrane-localized using antibody reagents, and the nature of their membrane attachment has been characterized. The comG operon encodes seven proteins which appear to be required for the binding of DNA to the cell surface. The comE and comF operons encode at least two proteins which are required for the uptake but not the binding of DNA. The ComFI protein resembles known DNA translocases and helicases, and may utilize the energy of ATP hydrolysis to drive DNA uptake. The ComE3 protein is suggested to be a pore-forming protein. We will use combined genetic, biochemical, immunological, electrophysiological and high resolution electron microscopic approaches to explore the arrangements of these proteins on the cell surface, their interrelationships and their functional roles during transformation. We will use cross-linking to detect proteins that contact DNA during transformation. We will investigate the polarity of DNA uptake, the role of the Competence.specific nuclease activity, and we will seek additional competence gene products. Finally we will attempt to establish an in vitro DNA transport system.
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