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The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall‐deficient S‐cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo‐electron tomography and live cell imaging to further characterize S‐cell extrusion in  Kitasatospora viridifaciens . We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S‐cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S‐cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament‐like protein FilP. Furthermore, micro‐aerobic culturing promotes the formation of S‐cells in the wild type, but the limited oxygen still impedes S‐cell formation in the Δ filP  mutant. These results demonstrate that S‐cell formation is stimulated by oxygen‐limiting conditions and dependent on functional cytoskeleton remodeling.

Formation of wall‐less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen‐limiting conditions