Bacterial cell wall-degrading enzymes induce basidiomycete natural product biosynthesis

Natural products play a vital role for intermicrobial interactions. In the basidiomycete arena an important representative is variegatic acid, a lactone natural product pigment whose ecological relevance stems from both inhibiting bacterial swarming and from indirect participation in breakdown of organic matter by brown-rotting fungi. Previous work showed that the presence of bacteria stimulates variegatic acid production. However, the actual external molecular trigger that prompts its biosynthesis in the mushroom hyphae remained unknown. Here, we report on the identification of Bacillus subtilis subtilisin E (AprE) and chitosanase (Csn) as primary inducers of pulvinic acid pigment formation. Using the established co-culture system of B. subtilis and Serpula lacrymans, we used activity-guided FPLC-based fractionation of B. subtilis culture supernatants and subsequent peptide fingerprinting to identify candidates, and their role was corroborated by means of a pigment production assay using heterologously produced chitosanase and subtilisin. B. subtilis mutants defective in either the aprE or the csn gene still triggered pigmentation, yet to a lower degree, which points to a multicausal scenario and suggests the combined activity of these cell wall polymer-attacking enzymes as true stimulus.