Visualisierung der DNA Rekombination in kompetenten Bakterien

感受态描述了来自各种不同物种的细菌细胞从其环境中摄取DNA并通过同源重组（HR）将DNA整合到其染色体中的生理状态。通过这种方式，细胞可以获得新的遗传信息，例如抗性基因，这是一个临床相关的问题。我们已经发现两种DNA重组蛋白RecA和RecN在感受态枯草芽孢杆菌细胞中的单个细胞极积累，DNA摄取机制位于该细胞极。我们已经表明，RecN是一种ATP依赖性的ssDNA结合蛋白，它与进入的ssDNA结合。RecA从这个极点形成短暂的快速生长的细丝，从极点延伸并将传入的DNA运输到染色体上的同源区域。因此，感受态是一个空间上高度组织化的过程，其中DNA从一极到染色体采取确定的路径。本研究的目的是可视化进一步胞质重组蛋白，以获得一个完整的图片的参与和时间顺序的功能的蛋白质介导的HR在能力。新的蛋白质也将被纯化和生物化学研究。将在已知感受态基因的各种缺失突变体中监测胞质蛋白的定位，以阐明定位的遗传要求。将使用FRET和BiFC监测蛋白质相互作用。

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.