Fig. 6.

Hyperosmotic stress conditions are sufficient to isolate L-form strains. a Phenotypic differences are evident when S-cells, penicillin-induced L-forms and cells of the M1 and M2 strains are grown on LPMA medium without (top) and with penicillin (PenG, bottom). Plating S-cells led to the formation of compact mycelial colonies on LPMA medium contrary to the other strains that formed viscous colonies. The addition of 0.6 mg ml⁻¹ penicillin abolished efficient switching of S-cells to the mycelial mode-of-growth. Small mycelial colonies derived from S-cells were only formed in the least diluted part (indicated with a dashed ellipse). In contrast, the addition of penicillin neither effected growth of the penicillin-induced L-forms, nor that of strains M1 and M2. b All cell wall-deficient cells were able to form mycelial colonies on MYM medium lacking high levels of osmolytes. Unlike the majority of colonies derived from S-cells, the penicillin-induced L-forms and strains M1 and M2 exclusively formed (brown) non-sporulating colonies. c Morphology of S-cells in comparison to cells of the penicillin-induced L-form strain and strains M1 and M2 grown for 48 h in LPB medium. Cells were stained with FM5-95 and SYTO-9 to visualize membranes and DNA, respectively. The arrowheads indicate intracellular vesicles, while empty vesicles are indicated with an asterisk. The inlay in M2 shows a proliferation-associated tubulation event. d–f Frames from time-lapse microscopy show L-form-like proliferation involving (d) vesiculation, (e) blebbing and (f) membrane tubulation. g Formation of S-cells upon prolonged exposure to hyperosmotic stress. Germination of spores under hyperosmotic stress conditions generates germlings, which are able to extrude S-cells. These S-cells are able to switch to the mycelial mode-of-growth, or sporadically acquire mutations that allow them to proliferate like L-forms, which is characterized by tubulation, blebbing or vesiculation. Scale bar represents 10 µm (c), 2 µm (inlay panel c) or 5 µm (d–f)