Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie, B6a, B-4000 Sart-Tilman, Belgium.
Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 1098 XH Amsterdam, The Netherlands.
Microbiology (Reading). 2011 Jan;157(Pt 1):251-259. doi: 10.1099/mic.0.040071-0. Epub 2010 Sep 16.
During the cell cycle of rod-shaped bacteria, two morphogenetic processes can be discriminated: length growth of the cylindrical part of the cell and cell division by formation of two new cell poles. The morphogenetic protein complex responsible for the septation during cell division (the divisome) includes class A and class B penicillin-binding proteins (PBPs). In Escherichia coli, the class B PBP3 is specific for septal peptidoglycan synthesis. It requires the putative lipid II flippase FtsW for its localization at the division site and is necessary for the midcell localization of the class A PBP1B. In this work we show direct interactions between FtsW and PBP3 in vivo and in vitro by FRET (Förster resonance energy transfer) and co-immunoprecipitation experiments. These proteins are able to form a discrete complex independently of the other cell-division proteins. The K2-V42 peptide of PBP3 containing the membrane-spanning sequence is a structural determinant sufficient for interaction with FtsW and for PBP3 dimerization. By using a two-hybrid assay, the class A PBP1B was shown to interact with FtsW. However, it could not be detected in the immunoprecipitated FtsW-PBP3 complex. The periplasmic loop 9/10 of FtsW appeared to be involved in the interaction with both PBP1B and PBP3. It might play an important role in the positioning of these proteins within the divisome.
在杆状细菌的细胞周期中，可以区分出两个形态发生过程：圆柱状细胞部分的长度生长和通过形成两个新的细胞极进行细胞分裂。负责细胞分裂过程中隔膜形成的形态发生蛋白复合物（divisome）包括 A 类和 B 类青霉素结合蛋白（PBPs）。在大肠杆菌中，B 类 PBP3 是隔膜肽聚糖合成的特异性蛋白。它需要假定的脂质 II 翻转酶 FtsW 定位在分裂部位，并且对于 A 类 PBP1B 在中隔的定位是必需的。在这项工作中，我们通过 FRET（Förster 共振能量转移）和共免疫沉淀实验显示了 FtsW 和 PBP3 之间在体内和体外的直接相互作用。这些蛋白质能够独立于其他细胞分裂蛋白形成离散的复合物。含有跨膜序列的 PBP3 的 K2-V42 肽是与 FtsW 相互作用和 PBP3 二聚化的结构决定因素。通过使用双杂交测定，显示 A 类 PBP1B 与 FtsW 相互作用。然而，它不能在免疫沉淀的 FtsW-PBP3 复合物中检测到。FtsW 的周质环 9/10 似乎参与了与 PBP1B 和 PBP3 的相互作用。它可能在这些蛋白质在分裂体中的定位中发挥重要作用。
