Class E sortase SrtE and two SrtE-dependent cell wall-anchored hydrophobic proteins are involved in morphogenesis in Actinoplanes missouriensis : occurrence of exploratory growth beyond genus Streptomyces

The actinomycete Actinoplanes missouriensis grows by extending branched hyphae and forms terminal sporangia containing a few hundred spores. A sortase-dependent spore surface protein, SspA, has recently been shown to be important for sporangium maturation in A. missouriensis . In this study, we functionally characterized all five sortase candidates in A. missouriensis and revealed that only a class E sortase (SrtE) is required for sporangium formation. The srtE null mutant strain scarcely produced sporangia, in contrast to the sspA null mutant, which produced fragile sporangia, indicating that there are other SrtE substrate proteins besides SspA. Unexpectedly, Δ srtE colonies displayed a significantly increased rate of surface area expansion on nutrient-rich solid media compared with wild-type colonies. The growing hyphae of the Δ srtE strain raised the pH of the surrounding environment, possibly by emitting basic volatile compounds. This growth mode shares several defining characteristics with the exploratory growth (or exploration) of Streptomyces . We identified two SrtE-dependent cell wall-anchored proteins (CwpA and CwpB) required for the suppression of latent exploration in the wild-type strain; colonies of single-gene mutants of cwpA and cwpB showed exploration. While the Δ cwpA strain formed normal sporangia, severe defects in the initiation of sporangium formation were observed in the Δ cwp B strain, indicating that CwpB, but not CwpA, is essential for sporangium formation. Based on these results, we propose that SrtE suppresses latent exploration and promotes the initiation of sporangium formation by making the cell surface hydrophobic through the attachment of the hydrophobic proteins CwpA and CwpB to the cell wall. IMPORTANCE Exploration has recently been established as a new growth mode in the filamentous actinobacterial genus Streptomyces , where rapidly expanding mycelia lead to extensive surface colonization on nutrient-rich solid media. However, the phylogenetic prevalence and genetic basis of this unique growth mode remain unclear. Here, we report the discovery of exploration in Actinoplanes missouriensis , a filamentous actinomycete that forms terminal sporangia and zoospores. Through extensive genetic experiments, we demonstrated that a class E sortase suppresses latent exploration via covalent anchoring of two surface hydrophobic proteins to the cell wall in A. missouriensis . This study revealed that exploration capabilities are prevalent among filamentous actinomycetes beyond Streptomyces and that the hydrophobic nature of the cell surface appears to be crucial for suppressing latent exploration in A. missouriensis . The hydrophobic nature of the cell surface also appears to be important for initiating sporangium formation on nutrient-poor solid media in A. missouriensis .