Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

Abstract

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. A key event in the bacterial cell cycle is the assembly of cytoskeletal proteins into a ring that defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the membrane and generate budding vesicles, a phenotype that has not been reported before. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results provide new insights on the self-organization of basic cytoskeletal elements involved in bacterial division. Moreover, they demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.