Unveiling Concealed Functions of Endosymbiotic Bacteria Harbored in the Ascomycete Stachylidium bicolor

Among the plethora of unusual secondary metabolites isolated from Stachylidium bicolor are the tetrapeptidic endolides A and B. Both tetrapeptides contain 3-(3-furyl)-alanine residues, previously proposed to originate from bacterial metabolism. Inspired by this observation, we aimed to identify the presence of endosymbiotic bacteria in S. bicolor and to discover the true producer of the endolides. The endobacterium Burkholderia contaminans was initially detected by 16S rRNA gene amplicon sequencing from the fungal metagenome and was subsequently isolated. It was confirmed that the tetrapeptides were produced by the axenic B. contaminans only when in latency. Fungal colonies unable to produce conidia and the tetrapeptides were isolated and confirmed to be free of B. contaminans A second endosymbiont identified as related to Sphingomonas leidyi was also isolated. In situ imaging of the mycelium supported an endosymbiotic relationship between S. bicolor and the two endobacteria. Besides the technical novelty, our in situ analyses revealed that the two endobacteria are compartmentalized in defined fungal cells, prevailing mostly in latency when in symbiosis. Within the emerging field of intracellular bacterial symbioses, fungi are the least studied eukaryotic hosts. Our study further supports the Fungi as a valuable model for understanding endobacterial symbioses in eukaryotes.IMPORTANCE The discovery of two bacterial endosymbionts harbored in Stachylidium bicolor mycelium, Burkholderia contaminans and Sphingomonas leidyi, is described here. Production of tetrapeptides inside the mycelium is ensured by B. contaminans, and fungal sporulation is influenced by the endosymbionts. Here, we illustrate the bacterial endosymbiotic origin of secondary metabolites in an Ascomycota host.