Radek, Renate

Summary Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of ‘ Candidatus Adiutrix intracellularis’, an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis . It represents a novel, deep‐branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont ‘ Candidatus Desulfovibrio trichonymphae’ in the anterior part of the host cell. The draft genome of ‘ Ca . Adiutrix intracellularis’ obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood–Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase‐linked formate dehydrogenases (FDH H ) and all other components of the recently described hydrogen‐dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO 2 and H 2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.

Summary The bacterial tree contains many deep‐rooting clades without any cultured representatives. One such clade is ‘ E ndomicrobia’, a class‐level lineage in the phylum E lusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free‐living member of this clade from sterile‐filtered gut homogenate of defaunated (starch‐fed) R eticulitermes santonensis . Strain R sa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO 2 . Ultrastructural analysis revealed a G ram‐negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of G roup IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and 15 N 2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to ‘ C andidatus   E ndomicrobium trichonymphae’ and other obligate endosymbionts, we propose the name ‘ E ndomicrobium proavitum ’ gen. nov., sp. nov. for the first isolate of this lineage and the name ‘ E ndomicrobia ’ class. nov. for the entire clade.

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.