Lack of a putative intrinsically disordered protein anchored to the sporangiospore cell wall causes fragile sporangium formation in Actinoplanes missouriensis

The filamentous actinomycete Actinoplanes missouriensis develops terminal sporangia on substrate mycelia via short sporangiophores. Each sporangium, surrounded by an outer envelope, contains a few hundred spores encapsulated by the sporangium matrix. In this study, we identified a spore surface-displayed protein, SspA, that is required for the structural strength of sporangia in A. missouriensis through suppressor screening using a spore release-deficient mutant. SspA has a sortase-dependent cell wall-localizing signal, and its mature part is predicted to be a putative intrinsically disordered protein. An sspA null mutant (Δ sspA ) strain formed sporangia, but the mutant sporangia were highly fragile and collapsed immediately to release spores when suspended in aquatic solutions. Transmission electron microscopy revealed that the Δ sspA sporangia did not mature normally; the electron-dense sporangium matrix was not observed in the peripheral region of each spore, and the outer envelope of some sporangia was damaged. Peptide-tagged SspA proteins produced in the Δ sspA strain were detected on the surface of the zoospores using the HiBiT system. The heat tolerance of Δ sspA zoospores was higher than that of wild-type zoospores, suggesting that SspA influences the frequency of cross-bridges in the cell wall peptidoglycan. Phenotypic changes in the Δ sspA strain were restored by introducing sspA with its own promoter into the Δ sspA strain. These results demonstrate that SspA is a sporangiospore cell wall-anchored protein required for the formation of rigid sporangium structures in A. missouriensis . It is speculated that SspA is involved in the production of the sporangium matrix polysaccharides.