Reproducible switching between a walled and cell wall-deficient lifestyle of actinomycetes using gradient agar plates

The cell wall is a shape-defining structure that envelopes almost all bacteria, protecting them from biotic and abiotic stresses. Paradoxically, some filamentous actinomycetes have a natural ability to shed their cell wall under influence of hyperosmotic stress. These wall-deficient cells can revert to their walled state when transferred to a medium without osmoprotection but often lyse due to their fragile nature. Here, we designed plates with an osmolyte gradient to reduce cell lysis and thereby facilitating the transition between a walled and wall-deficient state. These gradient plates allow determining of the osmolyte concentration where switching takes place, thereby enabling careful and reproducible comparison between mutants affected by switching. Exploring these transitions could give valuable insights into the ecology of actinomycetes and their biotechnological applications. HIGHLIGHTS- Using agar plates with a gradient of osmoprotectants, revertant Streptomycetes can gradually revert to a walled state, thereby dramatically decreasing lysis. - Our method allows precise determination of the osmolyte concentration where reversion takes place, allowing careful and reproducible comparison between mutants. - Gradient agar plates can also be used to study chemical differentiation in Streptomycetes as a response to osmotic stress.