In vitro reconstitution of Escherichia coli divisome activation

FtsA is crucial for assembly of the E. coli divisome, as it dynamically links cytoplasmic FtsZ filaments with transmembrane cell division proteins. FtsA allegedly initiates cell division by switching from an inactive polymeric to an active monomeric confirmation, which recruits downstream proteins and stabilizes FtsZ filaments. Here, we use biochemical reconstitution experiments combined with quantitative fluorescence microscopy to study divisome activation in vitro. We compare wildtype-FtsA with FtsA-R286W, a constantly active gain-of-function mutant and find that R286W outperforms the wildtype protein in replicating FtsZ treadmilling dynamics, stabilizing FtsZ filaments and recruiting FtsN. We attribute these differences to a faster membrane exchange of FtsA-R286W and its higher packing density below FtsZ filaments. Using FRET microscopy, we find that FtsN binding does not compete with, but promotes FtsA self-interaction. Our findings suggest a model where FtsA always forms dynamic polymers on the membrane, which re-organize during assembly and activation of the divisome.

FtsA对于大肠杆菌分裂复合体的组装至关重要，因其能动态地将细胞质中的FtsZ丝状结构与其跨膜细胞分裂蛋白相连接。据称，FtsA通过从无活性多聚体形态转变为活性单体形态来启动细胞分裂，这一转变能够招募下游蛋白并稳定FtsZ丝状结构。在本研究中，我们通过生物化学重组成实验与定量荧光显微镜相结合的方法，在体外研究了分裂复合体的激活过程。我们将野生型FtsA与常活性功能获得性突变体FtsA-R286W进行比较，发现R286W在复制FtsZ treadmilling动态、稳定FtsZ丝状结构以及招募FtsN方面优于野生型蛋白。我们将这些差异归因于FtsA-R286W在膜上的交换速度更快以及其位于FtsZ丝状结构下方的包装密度更高。利用FRET显微镜，我们发现FtsN的结合并不与FtsA的自我相互作用竞争，反而促进其发生。我们的研究结果表明，FtsA始终在膜上形成动态的多聚体，这些多聚体在分裂复合体的组装和激活过程中会重新组织。