Visualisierung der DNA Rekombination in kompetenten Bakterien

能力描述了不同物种的细菌细胞从环境中提取DNA并通过同源重组(HR)将DNA整合到染色体上的生理状态。通过这种方式，细胞可以获得新的遗传信息，例如与临床相关的抗药性基因。我们发现两种DNA重组蛋白RecA和RecN在感受态枯草杆菌细胞中积累在一个细胞极上，DNA摄取机制就在那里。我们已经证明RecN是一种依赖于ATP的单链DNA结合蛋白，可以与传入的单链DNA结合。从这个极点，RecA形成瞬间的快速生长的细丝，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，能力是一个在空间上高度组织的过程，在这个过程中，DNA从一极到染色体采取一条确定的路径。这项研究的目的是进一步可视化胞质重组蛋白，以获得参与HR的蛋白质在感受态过程中的参与和功能的时间顺序的完整图景。新的蛋白质也将被提纯并进行生化研究。胞浆蛋白的定位将在已知能力基因的各种缺失突变体中进行监测，以阐明定位的遗传要求。蛋白质相互作用将使用FRET和BIFC进行监测。

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.