Synthetic Assembly of Bacterial Divisome Machinery in Vitro

Abstract

Bacterial cells grow and reproduce through coordination of many proteins collectively known as the cell division machinery. This machinery facilitates the division and physical separation of one cell into two identical daughter cells. The components of this cell division machinery are similar between bacterial taxa. In Escherichia coli, the cytoskeletal protein FtsZ promotes the formation of a division septum at the cell center and assembles into a large protein structure called the Z‐ring. FtsZ protomers in the Z‐ring form large linear polymers that assemble in a head to tail arrangement. Protein interactions between FtsZ in the Z‐ring and additional cell division proteins direct peptidoglycan synthesis and facilitate septation of the cell. FtsN, which may trigger septation, is a bitopic membrane protein with a small cytoplasmic domain and large periplasmic domain containing a peptidoglycan‐binding SPOR domain. FtsN interacts with other cell division proteins on both sides of the cytoplasmic membrane. In the cytoplasm, FtsN interacts with FtsA, an actin homolog, which tethers FtsZ polymers to the membrane. In the periplasm FtsN interacts with the FtsQBL complex and may help to recruit peptidoglycan synthetases such as FtsI. One proposed linear mechanism for activating cell wall synthesis begins with FtsA, which binds to FtsN in the cytoplasm, and causes FtsN to relay information to proteins in the periplasm, such as FtsQBL, to begin remodeling of the cell wall. Thus, it is critical that FtsN localizes to the future site of septation in order to carry out this function. Here we use purified proteins, including FtsZ, FtsA, and FtsN, to reconstitute the membrane‐spanning division complex in vitro and assemble a synthetic divisome scaffold. This reconstituted divisome subassembly allows us to probe direct protein interactions, protein conformations, assembly architectures, and nucleotide requirements in the cell division pathway, thus providing novel insight into the mechanics of division.