Life without a wall or division machine in Bacillus subtilis

The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis . Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated. Life without walls Bacteria depend on a cell wall for protection against physical and chemical damage and osmotic lysis. But rare L-form cells, thought to lack a cell wall, have been observed in some bacterial species. They may play a role in avoiding recognition by host immune systems and antibiotics such as the penicillins that target cell wall synthesis. But L-form cells are extremely difficult to grow and manipulate, hence difficult to study. This could change with the development of a route to genetically tractable L-form cells of the common model organism, Bacillus subtilis . Early findings with this new system are the confirmation that no cell wall synthesis is needed for L-form cells to replicate, and that the normal cell division machinery is not required. Instead, L-forms use an extrusion-resolution mechanism to proliferate, forming multiple progeny in a single step. These findings could have evolutionary implications. The cell wall is found throughout the bacterial sub-kingdom, so was probably present in the last common ancestor of these ancient organisms. The novel replication process used by L-form cells could provide a window into modes of proliferation used before the advent of the cell wall. L-form cells can derive from various bacterial species and do not possess a cell wall. It is shown that Bacillus subtilis can convert