肌动蛋白样FtsA蛋白对细胞分裂机制的功能至关重要,或者分裂,在许多细菌中,包括大肠杆菌。先前的体外研究表明,纯化的野生型FtsA在脂质膜上组装成闭合的小环,但是FtsA的寡聚变体,如FtsAR286W和FtsAG50E可以绕过某些分裂缺陷,形成弧形和双链(DS)寡聚状态,分别,这可能反映了FtsA的非活性形式向活性形式的转化。然而,FtsA的哪种寡聚形式负责组装和激活分裂体仍未得到证实。这里,我们对FtsADS丝进行了体内交联分析,以表明它们在很大程度上取决于适当的分裂体组装,并且在细胞分裂的后期很普遍。我们还使用了以前报道的变体,该变体无法组装DS细丝,FtsAM96ER153D,研究FtsA寡聚状态在分裂体组装和激活中的作用。我们表明FtsAM96ER153D不能在体内形成DS丝,无法替换本机FTSA,并赋予显性阴性表型,强调DS灯丝阶段对FtsA功能的重要性。令人惊讶的是,然而,通过ftsL*或ftsW*超裂变等位基因激活分裂体抑制了显性阴性表型,并挽救了FtsAM96ER153D的功能。我们的结果表明,FtsADS细丝一旦组装就需要用于分裂体激活,但是它们对于分隔体组装或引导隔膜合成不是必需的。IMPORTANCECell分裂是细胞复制的基础。在像大肠杆菌这样的简单细胞中,肌动蛋白同源物FtsA对于细胞分裂至关重要,并在细胞质膜上组装成多种蛋白质丝。这些细丝不仅在细胞分裂的早期阶段帮助将微管蛋白样FtsZ的聚合物束缚到膜上,而且在将其他细胞分裂蛋白招募到称为分裂体的复合物中发挥关键作用。一旦组装好,大肠杆菌分裂体随后激活分裂隔膜的合成,将细胞一分为二。最近发现的一种FtsA的寡聚构象是反平行双链丝。结合体内交联和遗传学,我们提供的证据表明,这些FtsA双丝在激活隔膜合成酶中起着至关重要的作用。
The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. However, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here, we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionality of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.IMPORTANCECell division is fundamental for cellular duplication. In simple cells like Escherichia coli bacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments not only help tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, the E. coli divisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double-stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.