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D ow nloaded from 2 Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein (ALP), 15 ParM, segregates DNA by pushing two plasmids in opposite directions and forms the cur-rent paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and dis-assembly. It is unclear whether dynamic instability is an essential feature of all ALP-based segregation mechanisms or whether bacterial filaments can segregate plasmids by 20 different mechanisms. We expressed and purified AlfA, a plasmid-segregating ALP from Bacillus subtilis, and find that it forms filaments with a unique structure and biochemis-try: AlfA nucleates rapidly; polymerizes in ATP or GTP; and forms highly twisted, rib