Robichon Carine, King Glenn F, Goehring Nathan W, Beckwith Jon
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
J Bacteriol. 2008 Sep;190(18):6048-59. doi: 10.1128/JB.00462-08. Epub 2008 Jul 11.
Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the "bait") to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the "prey") to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.
细菌细胞分裂由一组组装形成称为分裂体的大型多蛋白复合物的蛋白质介导。最近对枯草芽孢杆菌和大肠杆菌的研究表明，细胞分裂蛋白参与多种协同结合相互作用，因此对这些相互作用的分析提出了技术挑战。我们在此报告使用大肠杆菌人工隔膜靶向系统来检测枯草芽孢杆菌细胞分裂蛋白DivIB、FtsL、DivIC和PBP 2B之间的相互作用。该技术涉及将其中一种蛋白质（“诱饵”）与靶向细胞中部的大肠杆菌蛋白质ZapA融合，并将第二个潜在相互作用的伙伴（“猎物”）与绿色荧光蛋白（GFP）融合。两种测试蛋白在大肠杆菌中的阳性相互作用导致GFP融合构建体的隔膜定位，这可以通过荧光显微镜检测到。使用该系统，我们提供了证据表明在大肠杆菌中枯草芽孢杆菌分裂体蛋白之间存在两组强蛋白质 - 蛋白质相互作用，即DivIC与FtsL以及DivIB与PBP 2B，它们独立于其他枯草芽孢杆菌细胞分裂蛋白，并且不会干扰宿主细胞中的胞质分裂过程。我们基于这四种枯草芽孢杆菌细胞分裂蛋白中三种或四种的共表达的研究表明，与之前的观点相反，这四种蛋白质之间的相互作用不够强，不足以在大肠杆菌中形成稳定的四蛋白复合物。最后，我们的结果表明，大肠杆菌人工隔膜靶向是一种用于检测和表征来自其他微生物的多蛋白复合物内稳定蛋白质 - 蛋白质相互作用的有效且替代的方法。我们方法的一个显著特征是它可能只检测到最强的相互作用，从而表明其他技术所暗示的一些相互作用可能要么相当弱要么是假阳性。
