Visualisierung der DNA Rekombination in kompetenten Bakterien

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Competence describes the physiological state in which bacterial cells from various different species take up DNA from their environment and incorporate the DNA into their chromosome via homologous recombination (HR). This way, the cell can gain novel genetic information, e.g. resistance genes, a clinically relevant problem. We have found that two DNA recombination proteins, RecA and RecN, accumulate at a single cell pole in competent Bacillus subtilis cells, where the DNA uptake machinery is located. We have shown that RecN is an ATP dependent ssDNA-binding protein that binds to incoming ssDNA. From this pole, RecA forms transient fast-growing filaments that extend from the pole and transport the incoming DNA to the homologous region on the chromosome. Thus, competence is a spatially highly organized process, in which DNA takes a defined path from one pole to the chromosome. The aim of this study is to visualize further cytosolic recombination proteins, to obtain a complete picture of the involvement and temporal order of functions of proteins mediating HR during competence. Novel proteins will also be purified and studied biochemically. Localization of cytosolic proteins will be monitored in various deletion mutants in known competence genes to elucidate genetic requirements for localization. Protein interactions will be monitored using FRET and BiFC.