Genome analyses of uncultured TG2/ZB3 bacteria in 'Margulisbacteria' specifically attached to ectosymbiotic spirochetes of protists in the termite gut.

We investigated the phylogenetic diversity, localisation and metabolism of an uncultured bacterial clade, Termite Group 2 (TG2), or ZB3, in the termite gut, which belongs to the candidate phylum 'Margulisbacteria'. We performed 16S rRNA amplicon sequencing analysis and detected TG2/ZB3 sequences in 40 out of 72 termite and cockroach species, which exclusively constituted a monophyletic cluster in the TG2/ZB3 clade. Fluorescence in situ hybridisation analysis in lower termites revealed that these bacteria are specifically attached to ectosymbiotic spirochetes of oxymonad gut protists. Draft genomes of four TG2/ZB3 phylotypes from a small number of bacterial cells were reconstructed, and functional genome analysis suggested that these bacteria hydrolyse and ferment cellulose/cellobiose to H2, CO2, acetate and ethanol. We also assembled a draft genome for a partner Treponema spirochete and found that it encoded genes for reductive acetogenesis from H2 and CO2. We hypothesise that the TG2/ZB3 bacteria we report here are commensal or mutualistic symbionts of the spirochetes, exploiting the spirochetes as H2 sinks. For these bacteria, we propose a novel genus, 'Candidatus Termititenax', which represents a hitherto uncharacterised class-level clade in 'Margulisbacteria'. Our findings add another layer, i.e., cellular association between bacteria, to the multi-layered symbiotic system in the termite gut.

Phylogenetic Diversity and Single-Cell Genome Analysis of "Melainabacteria", a Non-Photosynthetic Cyanobacterial Group, in the Termite Gut.

Termite guts harbor diverse yet-uncultured bacteria, including a non-photosynthetic cyanobacterial group, the class "Melainabacteria". We herein reported the phylogenetic diversity of "Melainabacteria" in the guts of diverse termites and conducted a single-cell genome analysis of a melainabacterium obtained from the gut of the termite Termes propinquus. We performed amplicon sequencing of 16S rRNA genes from the guts of 60 termite and eight cockroach species, and detected melainabacterial sequences in 48 out of the 68 insect species, albeit with low abundances (0.02-1.90%). Most of the melainabacterial sequences obtained were assigned to the order "Gastranaerophilales" and appeared to form clusters unique to termites and cockroaches. A single-cell genome of a melainabacterium, designated phylotype Tpq-Mel-01, was obtained using a fluorescence-activated cell sorter and whole genome amplification. The genome shared basic features with other melainabacterial genomes previously reconstructed from the metagenomes of human and koala feces. The bacterium had a small genome (~1.6 Mb) and possessed fermentative pathways possibly using sugars and chitobiose as carbon and energy sources, while the pathways for photosynthesis and carbon fixation were not found. The genome contained genes for flagellar components and chemotaxis; therefore, the bacterium is likely motile. A fluorescence in situ hybridization analysis showed that the cells of Tpq-Mel-01 and/or its close relatives are short rods with the dimensions of 1.1±0.2 µm by 0.5±0.1 µm; for these bacteria, we propose the novel species, "Candidatus Gastranaerophilus termiticola". Our results provide fundamental information on "Melainabacteria" in the termite gut and expand our knowledge on this underrepresented, non-photosynthetic cyanobacterial group.

Discovery of ectosymbiotic Endomicrobium lineages associated with protists in the gut of stolotermitid termites.

The genus Endomicrobium is a dominant bacterial group in the gut of lower termites, and most phylotypes are intracellular symbionts of gut protists. Here we report the discovery of Endomicrobium ectosymbionts of termite gut protists. We found that bristle-like Endomicrobium cells attached to the surface of spirotrichosomid protist cells inhabiting the termite Stolotermes victoriensis. Transmission electron microscopy revealed that a putative Endomicrobium cell likely attached to the protist surface via a protrusion from the tip of the bacterium. A phylotype, sharing 98.9% 16S rRNA sequence identity with the Endomicrobium ectosymbionts of the spirotrichosomid protists, was also found on the cell surface of the protist Trichonympha magna in the gut of the termite Porotermes adamsoni. We propose the novel species 'Candidatus Endomicrobium superficiale' for these bacteria. T. magna simultaneously harboured another Endomicrobium ectosymbiont that shared 93.5-94.2% 16S rRNA sequence identities with 'Ca. Endomicrobium superficiale'. Furthermore, Spirotrichonympha-like protists in P. adamsoni guts were associated with an Endomicrobium phylotype that possibly attached to the host flagella. A phylogenetic analysis suggested that these ectosymbiotic lineages have evolved multiple times from free-living Endomicrobium lineages and are relatively distant from the endosymbionts. Our results provide novel insights into the ecology and evolution of the Endomicrobium.