Genome segregation in bacteria: investigating protein dynamics and mechanisms in the cell at single molecule level

细菌耐多药是世界范围内人类健康的全球性负担。负责抗生素耐药性的大的、低拷贝数的质粒已经进化出策略来确保它们在细胞分裂时的忠实分布。多药耐药质粒拥有自己的生存系统，即分离盒，这确保了质粒在细胞分裂时的准确分离。当这个系统出现故障时，质粒不能稳定地遗传，最终会丢失，导致细菌对抗菌素敏感。多药耐药质粒TP228在大肠杆菌中以低拷贝数复制。它的分割盒编码两种蛋白质：ParF（一种atp酶）和ParG（一种与质粒上特定位点相关的dna结合蛋白）。通过在活细胞上使用超分辨率显微镜，我们已经表明，parg -质粒复合物被包裹在一个三维的ParF网络中，该网络通过细菌染色体的体积组装。当ParG蛋白有缺陷时，ParG-质粒复合物被排除在染色体体积之外，并在随后的细胞分裂中丢失。我们提出了一个捕蝇草模型作为质粒分离的机制(1)。该项目将研究parfg -质粒复合物在细胞中的定位以及单分子水平上复合物形成的动力学，以揭示质粒分离的机制细节。

Bacterial multidrug resistance is a global burden on human health worldwide.Large, low copy number plasmids responsible for antibiotic resistance haveevolved strategies to ensure their faithful distribution at celldivision. Multidrug resistance plasmids harbour their own survival system, apartition cassette, which ensures an accurate segregation of the plasmids atcell division. When this system malfunctions, the plasmid is not stablyinherited and is ultimately lost causing bacteria to become sensitive toantimicrobials. The multidrug resistance plasmid TP228 replicates at lowcopy number in Escherichia coli. Its partition cassette encodes two proteins:ParF, an ATPase, and ParG, a DNA-binding protein that associates to aspecific site on the plasmid. By using super-resolution microscopy on livecells, we have shown that ParG-plasmid complexes are entrapped within athree-dimensional ParF meshwork that assembles through the volume of thebacterial chromosome. When the ParG protein is defective, the ParG-plasmidcomplex is excluded from the chromosome volume and lost at the followingcell division. We have proposed a Venus flytrap model as a mechanism forplasmid segregation (1).This project will investigate the localization of ParFG-plasmid complexes inthe cell and the dynamics of complex formation at single molecule level toshed light on the mechanistic details underpinning plasmid segregation.