Endomicrobium proavitum, the first isolate of Endomicrobia class. nov. (phylum Elusimicrobia) – an ultramicrobacterium with an unusual cell cycle that fixes nitrogen with a Group IV nitrogenase

Abstract

The bacterial tree contains many deep-rooting clades without any cultured representatives. One such clade is 'Endomicrobia', a class-level lineage in the phylum Elusimicrobia 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) Reticulitermes santonensis. Strain Rsa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO2. Ultrastructural analysis revealed a Gram-negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of Group 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)N2 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 'Candidatus Endomicrobium trichonymphae' and other obligate endosymbionts, we propose the name 'Endomicrobium proavitum' gen. nov., sp. nov. for the first isolate of this lineage and the name 'Endomicrobia' class. nov. for the entire clade.

Potential for Nitrogen Fixation in the Fungus-Growing Termite Symbiosis

“…The high diversity of nifH genes or transcripts discovered in some of the above studies (Ohkuma et al, 1999; Yamada et al, 2007), and previous work, indicates that other gut bacterial taxa may also contribute (Potrikus and Breznak, 1977; Doolittle et al, 2008; Isanapong et al, 2012; Wertz et al, 2012; Zheng et al, 2016). In addition, the lower termites harbor unique flagellated protists in their guts that play a key role in host nutrition; these protists harbor bacterial endosymbionts (Hongoh et al, 2008; Ohkuma et al, 2015) or ectosymbionts (Desai and Brune, 2012) that may also fix N 2 .…”

“…Similarly, a previous study in Odontotermes formosanus found only few functional nifH genes compared to those found in wood-eating termites (Yamada et al, 2007), and these were believed to belong to a “pseudo” nifH clade (Ohkuma et al, 1999). The latter finding has, however, been challenged by recent work (Zheng et al, 2016). Thus, targeted work to elucidate the presence and expression of nifH and the responsible producers is needed.…”

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches

“…S4 and S5 in the supplemental material). Together, these proteins were distantly related to their homologues in Endomicrobium proavitum, an Elusimicrobia isolate from the hindguts of Reticulitermes santonensis recently shown to be linked to nitrogen fixation (71).…”

“…However, in termite guts and in Bacteroidales symbionts encoding homologues to both Fe-only and Mo-dependent NifH, Fe-only NifH (AnfH) was preferentially expressed despite providing additional molybdenum (35,37). Cluster IV NifH was not thought to contribute to nitrogen fixation, but Endomicrobium proavitum (belonging to the Endomicrobia and a freeliving termite hindgut microbe) fixes nitrogen while only encoding a cluster IV NifH, and also NifD-like and NifK-like proteins (71). Barbulanympha ectosymbionts have multiple, diverse nitrogenase operons, suggesting that the ectosymbionts have several means to achieve nitrogen fixation.…”

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

“…In particular, two sequences are part of recently identified phyla (Candidatus Cloacimonete and Elusimicrobia) that have no or very few cultivated representatives. 64 , 65 Overall, most of the species present in the Hyp enrichments are closely related to sequenced organisms that encode HypD, indicating they likely also possess this enzyme (Fig. 7).…”