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Three thermophilic Nanoarchaeota-Crenarchaeota symbiotic systems have been described. We obtained another stable anaerobic enrichment culture at 80°C, pH 6.0 from a New Zealand hot spring. The nanoarchaeote (Ncl-1) and its host (NZ3T) were isolated in co-culture and their genomes assembled. The small (~200 nm) flagellated cocci were often attached to larger cocci. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi. Their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Ncl-1 also likely relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3T was isolated and grows on proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3T requires thiosulfate and grows best at 82°C, pH 6.0. NZ3T is most closely related to the Desulfurococcaceae, Ignisphaera aggregans (~92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3T 32 represent novel genera in the Nanoarchaeota and Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3T (=DSMZ 107634T =OCM 1213T =JCM 32895T). Introduction The Archaea still remain the most unexplored of life's domains, despite a ubiquitous distribution and a diversity that may parallel that of the Bacteria. Many new lineages without cultivated representatives have been discovered through metagenomic and single cell genomic approaches, giving rise to a significant increase in new archaeal phyla and divisions [4,7,89,95,105]. One exception is the Nanoarchaeota, a phylum with only two representatives in culture [36,113]. The first isolate, Nanoarchaeum equitans, was obtained from a shallow marine hydrothermal site off Iceland and forms an obligate parasitic relationship with its autotrophic archaeal host, Ignicoccus hospitalis [36]. With its highly reduced genome, N. equitans lacks most primary biosynthetic functions and has to acquire many metabolic precursors from its host [111]. The second nanoarchaeote in culture was obtained from an acidic hot spring in Yellowstone National Park (YNP) [113]. This terrestrial representative, Candidatus Nanopusillus acidilobi, is also an obligate symbiont, and its host is an acidophile and heterotroph, Acidilobus sp. 7A. Like its marine relative, Cand. Nps. acidilobi shows extensive genome reduction and requires its host for most primary biosynthetic and energetic needs, yet it has retained some potential for glycolysis and gluconeogenesis, expresses archaeal flagella genes and lacks a membrane ATP synthase complex [113]. The differences between the two cultivated nanoarchaeotes point to how diverse symbiotic associations may be within the 33 Nanoarchaeota. Additional terrestrial Nanoarchaeota symbiotic systems were characterized based on single cell sequencing from geothermal acidic pools in YNP [72,89]. Even though these environmental nanoarchaeote genomes were very similar to Cand. Nps. acidilobi, their implied hosts were quite dissimilar and more closely related to Sulfolobus than Acidilobus [72,89]. Based on 16S rRNA gene surveys, Nanoarchaeota have been detected globally in terrestrial and marine hot springs. These include many sites in YNP [16,34,71,72], Kamchatka [15,34], Chile [14], Central Asia [14], China [14], New Zealand [14], and deep-sea vents in the Atlantic [24] and Pacific oceans [26,34,67,73]. These different nanoarchaeotes form phylogenetic clades that can be separated somewhat by geographical region. It is therefore likely that as more members of this group and their hosts are characterized, a much better understanding of their biology and evolutionary history will emerge. Here, we report on the enrichment and isolation of a third nanoarchaeote in stable co-culture with its host. This nanoarchaeote was obtained from a terrestrial hot spring in New Zealand and more closely resembles its relatives from YNP than N. equitans. We also describe the isolation and characterization of the host, a novel genus in the Desulfurococcaceae. The Nanoarchaeota-host system highlights the need for formal recognition and classification for organisms that can be cultivated, but only in the presence of a symbiotic partner. 34 Materials and methods Sample site and culturing conditions Samples from “Cooking Pots” hot springs at Hell’s Gate, Tikitere, New Zealand (38.0654°S, 176.3595°E) were inoculated into anaerobic enrichment media: Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) media #88 with (g/L): Na2O3S2.5H2O, 2.0; NaOH, 0.16; yeast extract, 0.5; tryptone, 0.5; resazurin, 0.001; FeCl3.6H2O, 0.004; MnCl2.4H20, 0.00036; and 10 mL additional trace mineral solution DSMZ #141 and dispensed under N2/CO2 (80/20, v/v) and pH adjusted to 6.0-6.1. The enrichments were incubated at 80°C for four days and checked by phase contrast microscopy. DNA was extracted using the Qiagen DNeasy Blood and Tissue DNA kit and the presence of Nanoarchaeota was checked using nanoarchaeotal-specific 16S rRNA gene primers [34]. Nanoarchaeotal-positive enrichment cultures were monitored over many transfers and maintained stably for over two years. A nanoarchaeote-host coculture was isolated after several dilution-to-extinction transfers. Subsequently, the coculture was transferred into, and routinely grown in, the above media with the following modifications: 0.5 g/L casein hydrolysate, 1.0 g/L yeast extract, 1.0 g/L tryptone, 0.0625 g/L L-cysteine-HCl and 10 mL/L glycerol. Host isolation and 16S rRNA gene sequencing The host was isolated by several series of dilution-to-extinction transfers in the described modified enrichment media. Genomic DNA was extracted as above, and the purity of the isolate was verified by PCR and sequencing of the 16S rRNA gene. Due to the presence of introns in the host 16S rRNA gene, two overlapping primer sets were 35 utilized for sequencing: 4F (5’-TCCGGTTGATCCTGCCRG-3’), 907RN (5’CCGYCAATTCMTTTRAGTTT-3’), 341FA (5’-CCTAYGGGGYGCASCAGGCG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). PCR conditions consisted of initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s, followed by a final elongation cycle at 72°C for 7 min. Physiological characterization of the host Unless otherwise noted, all characterization studies were carried out in triplicate at 80-85°C in sealed anaerobic Balch tubes. Cell density was monitored by phase contrast microscopy with a Petroff-Hausser counting chamber. Use of carbon sources was determined at a 0.1% (w/v) final concentration using a modified enrichment media with reduced yeast extract (0.01% w/v) and no casein hydrolysate, tryptone, or glycerol. Cultures were transferred at least once to minimize carry-over. The carbon sources that were tested included yeast extract, tryptone, casamino acids, gelatin, acetate, butyrate, cellulose, fructose, glucose, glycerol, mannose, propionate, sorbitol, ribose, starch, sucrose and xylose. Alternate electron acceptor use was determined by replacing L-cysteine-HCl and thiosulfate in the enrichment media with a variety of electron acceptors at both 2.5 and 10 mM final concentrations. To prevent sublimation, elemental sulfur (2.5 and 10 mM) was added directly to media in individual Balch tubes prior to autoclaving at 105°C for 1 hour [25]. Potential electron acceptors included elemental sulfur, nitrate, thiosulfate, sulfite and L-cysteine-HCl. A positive growth response was only recorded after three consecutive transfers in the same medium. 36 To assess the pH range of isolate growth, enrichment media was supplemented with 25 mM MES (pH 5.5-6.0) and 25 mM MOPS (pH 6.3-6.8) [78]. The pH was adjusted to the desired range prior to autoclaving. Oxygen tolerance was tested by adding defined volumes (0-10%, v/v) of filtered oxygen directly to anaerobic culture tubes containing media with 10 mM thiosulfate but lacking L-cysteine HCl. Requirement for NaCl was determined using 0-1% NaCl (w/v). Core and intact lipid preparation and analysis Lipids were recovered using a modified Bligh and Dyer method [9], and polar head groups were removed by acidic methanolysis [79]. Core and intact lipids were analyzed using a modified version of the protocol described by Elling et al, 2016 [22]. Liquid chromatography-mass spectrometry (LCMS) was performed using a Shimadzu 8040-LCMS system equipped with a photodiode array detector and a triple quadrupole low resolution MS utilizing an ESI source for ionization and Q3 scanning from 300 to 2000 m/z. Lipids were identified based on comparison with published mass spectrometry data [69,115]. Additional methodologies for core and intact polar lipid analysis are available in Appendix A.

Symbiosis in Archaea: Functional and Phylogenetic Diversity of Marine and Terrestrial Nanoarchaeota and their Hosts