Metabolic capacities of large “pillotinaceous” spirochetes from termite guts and their placement among Breznakiellaceae

BACKGROUND: Spirochetes are the most abundant bacterial group in the hindgut of termites. The largest species, with cell lengths of up to 100 µm, have been provisionally classified in the family "Pillotinaceae" based exclusively on morphological traits. However, in the absence of cultured representatives, their phylogenetic position and metabolism remain entirely unknown. RESULTS: We investigated phylogeny and metabolic capacities of "pillotinaceous" spirochetes using single-cell techniques, electron microscopy, and fluorescence in situ hybridization. All sequences of large spirochetes obtained from various termites fell into four distinct, well-supported clusters within the family Breznakiellaceae. Based on ultrastructural features, three of the clusters were assigned to the genera Pillotina, Hollandina, and the newly established genus Hollandinoides; a fourth cluster was tentatively assigned to the genus Clevelandina. Functional analysis of the single-cell genomes of Pillotina corrugata sp. nov., Hollandina grandis sp. nov., and Hollandinoides gharagozlouae gen. nov. sp. nov., combined with comparative genomics of other uncultured relatives, demonstrated differences in the capacity to degrade cellulose, hemicelluloses, and dextrins. While members of the genus Pillotina have a fermentative metabolism, members of the other genera encode a Wood-Ljungdahl pathway and, in the case of Hollandina, a group-III nitrogenase, suggesting roles in reductive acetogenesis and nitrogen fixation. CONCLUSIONS: Our results provide the first molecular data on pillotinaceous spirochetes. We show that the three genera covered in our study belong to the family Breznakiellaceae, which harbors the majority of termite-gut spirochetes. Comparative genome analysis indicated that the large spirochetes in termite guts have distinct roles in symbiotic digestion.