Treitli, Sebastian C.

Abstract 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.

Lower termites harbor in their hindgut complex microbial communities that are involved in the digestion of cellulose. Among these are protists, which are usually associated with specific bacterial symbionts found on their surface or inside their cells. While these form the foundations of a classic system in symbiosis research, we still know little about the functional basis for most of these relationships. Here, we describe the complex functional relationship between one protist, the oxymonad Streblomastix strix , and its ectosymbiotic bacterial community using single-cell genomics. We generated partial assemblies of the host S. strix genome and Candidatus Ordinivivax streblomastigis, as well as a complex metagenome assembly of at least 8 other Bacteroidetes bacteria confirmed by ribosomal (r)RNA fluorescence in situ hybridization (FISH) to be associated with S. strix. Our data suggest that S. strix is probably not involved in the cellulose digestion, but the bacterial community on its surface secretes a complex array of glycosyl hydrolases, providing them with the ability to degrade cellulose to monomers and fueling the metabolism of S. strix . In addition, some of the bacteria can fix nitrogen and can theoretically provide S. strix with essential amino acids and cofactors, which the protist cannot synthesize. On the contrary, most of the bacterial symbionts lack the essential glycolytic enzyme enolase, which may be overcome by the exchange of intermediates with S. strix . This study demonstrates the value of the combined single-cell (meta)genomic and FISH approach for studies of complicated symbiotic systems.