Bacillus endospore appendages form a novel family of disulfide-linked pili

is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here we isolate endospore appendages (Enas) from the food poisoning outbreak strain NVH 0075-95 and find proteinaceous fibers of two main morphologies. By using cryo-EM and 3D helical reconstruction we show that Enas form a novel class of Gram-positive pili. Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought and chemical damage. Phylogenomic analysis reveals the presence of defined clades amongst different eco- and pathotypes. We propose Enas to represent a novel class of pili specifically adapted to the harsh conditions encountered by bacterial spores.