Peptidoglycan-reshuffling proteins SCO0954, SCO1758, SCO4439, and SCO4440 modulate the formation of wall-deficient cells in Streptomyces coelicolor under hyperosmotic sucrose stress.

Streptomycetes are biotechnologically valuable bacteria with complex cell division that produce extracellular vesicles (EVs), typically nanometre-sized but can reach 2.5 μm in diameter. Streptomyces also produce dividing wall-deficient L-forms (0.5-7 μm diameter) and, under hyperosmotic stress, non-dividing wall-deficient S-cells (3-4 μm diameter). The boundaries between EVs, L-forms and S-cells are not always clear, as large DNA-containing EVs can resemble small L-forms and S-cells in size. Both EVs and wall-deficient cells offer competitive advantages, such as inter-bacterial signalling, antibiotic transport, resistance and phage defence. However, their formation mechanisms remain poorly understood. We identified sco1758 (engA GTPase), sco0954 (methionine N-acetyltransferase), sco4439 (D-Ala-D-Ala carboxypeptidase), and sco4440 (GOLPH3-like) as important for wall-deficient cell formation in Streptomyces coelicolor under hyperosmotic sucrose conditions. Mutations in sco4439 and sco4440 increased tetra-tetra(Gly) and tetra(Gly)-penta(Gly) (4-3) peptidoglycan (PG) dimers, while sco1758 affected only the former. Complementation reversed these changes. sco0954 overexpression enhanced PG-associated methionine acetylation and oxidation. Our findings suggest that PG dimerisation and methionine modification may contribute to the formation of wall-deficient cells under hyperosmotic sucrose stress. Further research is required to elucidate how SCO1758, SCO0954 and SCO4439/40 modulate PG architecture and to evaluate their potential to promote EV production for biotechnological applications.