AbstractThe ancient fungus-growing termite (Mactrotermitinae) symbiosis involves the obligate association between a lineage of higher termites and basidiomycete Termitomyces cultivar fungi. Our investigation of the fungus-growing termite Macrotermes natalensis shows that Bacillus strains from M. natalensis colonies produce a single major antibiotic, bacillaene A (1), which selectively inhibits known and putatively antagonistic fungi of Termitomyces. Comparative analyses of the genomes of symbiotic Bacillus strains revealed that they are phylogenetically closely related to Bacillus subtilis, their genomes have high homology with more than 90% of ORFs being 100% identical and the sequence identities across the biosynthetic gene cluster for bacillaene are higher between termite-associated strains than to the cluster previously reported in B. subtilis. Our findings suggest that this lineage of antibiotic-producing Bacillus may be a defensive symbiont involved in the protection of the fungus-growing termite cultivar.