A mesophilic, neutrophilic and aerobic, ammonia-oxidizing archaeon, strain EN76T, was isolated from garden soil in Vienna (Austria). Cells were irregular cocci with a diameter of 0.6–0.9 µm and possessed archaella and archaeal pili as cell appendages. Electron microscopy also indicated clearly discernible areas of high and low electron density, as well as tubule-like structures. Strain EN76T had an S-layer with p3 symmetry, so far only reported for members of the Sulfolobales. Crenarchaeol was the major core lipid. The organism gained energy by oxidizing ammonia to nitrite aerobically, thereby fixing CO2, but growth depended on the addition of small amounts of organic acids. The optimal growth temperature was 42 °C and the optimal pH was 7.5, with ammonium and pyruvate concentrations of 2.6 and 1 mM, respectively. The genome of strain EN76T had a DNA G+C content of 52.7 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain EN76T is affiliated with the recently proposed phylum Thaumarchaeota, sharing 85 % 16S rRNA gene sequence identity with the closest cultivated relative ‘Candidatus Nitrosopumilus maritimus’ SCM1, a marine ammonia-oxidizing archaeon, and a maximum of 81 % 16S rRNA gene sequence identity with members of the phyla Crenarchaeota and Euryarchaeota and any of the other recently proposed phyla (e.g. ‘Korarchaeota’ and ‘Aigarchaeota’). We propose the name Nitrososphaera viennensis gen. nov., sp. nov. to accommodate strain EN76T. The type strain of Nitrososphaera viennensis is strain EN76T ( = DSM 26422T = JMC 19564T). Additionally, we propose the family Nitrososphaeraceae fam. nov., the order Nitrososphaerales ord. nov. and the class Nitrososphaeria classis nov.

Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “Candidatus Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “Ca. Nitrotoga” culture was available. Here we obtained the first “Ca. Nitrotoga” isolate from activated sludge. This organism, “Candidatus Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “Ca. Nitrotoga” enrichments, which were described as cold-adapted NOB. “Ca. Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO2−) and nitrate (up to 25 mM NO3−). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “Ca. Nitrotoga fabula” were reconstructed from the closed genome. “Ca. Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “Ca. Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “Ca. Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “Ca. Nitrotoga fabula” to survive nitrite depletion and colonize new niches.

Harnessing chemosynthetic symbionts is a recurring evolutionary strategy. Eukaryotes from six phyla as well as one archaeon have acquired chemoautotrophic sulfur-oxidizing bacteria. In contrast to this broad host diversity, known bacterial partners apparently belong to two classes of bacteria-the Gamma-and Epsilonproteobacteria. Here, we characterize the intracellular endosymbionts of the mouthless catenulid flatworm genus Paracatenula as chemoautotrophic sulfur-oxidizing Alphaproteobacteria. The symbionts of Paracatenula galateia are provisionally classified as "Candidatus Riegeria galateiae" based on 16S ribosomal RNA sequencing confirmed by fluorescence in situ hybridization together with functional gene and sulfur metabolite evidence. 16S rRNA gene phylogenetic analysis shows that all 16 Paracatenula species examined harbor host species-specific intracellular Candidatus Riegeria bacteria that form a monophyletic group within the order Rhodospirillales. Comparing host and symbiont phylogenies reveals strict cocladogenesis and points to vertical transmission of the symbionts. Between 33% and 50% of the body volume of the various worm species is composed of bacterial symbionts, by far the highest proportion among all known endosymbiotic associations between bacteria and metazoans. This symbiosis, which likely originated more than 500 Mya during the early evolution of flatworms, is the oldest known animalchemoautotrophic bacteria association. The distant phylogenetic position of the symbionts compared with other mutualistic or parasitic Alphaproteobacteria promises to illuminate the common genetic predispositions that have allowed several members of this class to successfully colonize eukaryote cells. intracellular symbiosis | marine catenulid | meiofauna | subtidal sand

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3–6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host’s internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11–13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan’s nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

Rod-shaped bacteria usually grow in length and place their FtsZ ring and division site at midcell, perpendicular to their long axis [1,2]. Here, we provide morphometric and immunocytochemical evidence that a nematode-associated gammaproteobacterium [3,4] grows in width, sets a constricting FtsZ ring parallel to its long axis, and divides longitudinally by default. Remarkably, the newly described FtsZ ring appears to be not only 90° shifted with respect to model rods, but also elliptical and discontinuous. This reveals an unexpected versatility of the gammaproteobacterial cytokinetic machinery.

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1–3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, ‘Candidatus Celerinatantimonas neptuna’, that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.

Bathymodiolus mussels dominate animal communities at many hydrothermal vents and cold seeps. Essential to the mussels' ecological and evolutionary success is their association with symbiotic methane- and sulfur-oxidizing gammaproteobacteria, which provide them with nutrition. In addition to these well-known gammaproteobacterial endosymbionts, we found epsilonproteobacterial sequences in metatranscriptomes, metagenomes and 16S rRNA clone libraries as well as by polymerase chain reaction screening of Bathymodiolus species sampled from vents and seeps around the world. These epsilonproteobacterial sequences were closely related, indicating that the association is highly specific. The Bathymodiolus-associated epsilonproteobacterial 16S rRNA sequences were at most 87.6% identical to the closest cultured relative, and 91.2% identical to the closest sequences in public databases. This clade therefore represents a novel family within the Epsilonproteobacteria. Fluorescence in situ hybridization and transmission electron microscopy showed that the bacteria are filamentous epibionts associated with the gill epithelia in two Bathymodiolus species. In animals that host highly specific symbioses with one or a few types of endosymbionts, other less-abundant members of the microbiota can be easily overlooked. Our work highlights how widespread and specific associations with less-abundant microbes can be. Possibly, these microbes play an important role in the survival and health of their animal hosts.