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

Abstract

Abstract 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.