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

The filamentous actinomycete <i>Actinoplanes missouriensis</i> 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 <i>A. missouriensis</i> 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 <i>sspA</i> null mutant (Δ<i>sspA</i>) 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<i>sspA</i> 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<i>sspA</i> strain were detected on the surface of the zoospores using the HiBiT system. The heat tolerance of Δ<i>sspA</i> 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<i>sspA</i> strain were restored by introducing <i>sspA</i> with its own promoter into the Δ<i>sspA</i> strain. These results demonstrate that SspA is a sporangiospore cell wall-anchored protein required for the formation of rigid sporangium structures in <i>A. missouriensis</i>. It is speculated that SspA is involved in the production of the sporangium matrix polysaccharides.