Anaerobic endosymbiont generates energy for ciliate host by denitrification

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe ‘Candidatus Azoamicus ciliaticola’, which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. ‘Candidatus A. ciliaticola’ contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron–sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. ‘Candidatus A. ciliaticola’ and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis 1,2 . Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution 3 . As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation 4 . Here we describe 'Candidatus Azoamicus ciliaticola', which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. 'Candidatus A. ciliaticola' contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron-sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. 'Candidatus A. ciliaticola' and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.
Eukaryotic life evolved at least around 1-1.9 billion years ago 5,6 , after the gradual oxygenation of the atmosphere of the Earth (which started about 2.4 billion years ago 7,8 ). The ensuing diversification of eukaryotes has been attributed to their ability to harvest copious amounts of energy from the respiration of oxygen, an abundant, high-potential electron acceptor. Oxygen respiration is performed in mitochondria, which are specialized subcellular organelles that arose from a free-living prokaryotic organism through endosymbiosis 9 . During the evolution towards the contemporary organelle, the endosymbiont has either lost its genes or transferred them to the host genome; mitochondria have kept only a small subset of genes 10 that pertain to translation and the electron transport chain 11,12 . Some eukaryotes that inhabit anoxic environments do not contain aerobic mitochondria. Originally thought to be 'ancestral' 13 , all of these amitochondriate eukaryotes in fact evolved secondarily from predecessors that had aerobic mitochondria 14 ; they possess highly reduced mitochondria-related organelles or at least contain genes of mitochondrial origin in their nuclear genomes 15 . Throughout the course of evolution, anaerobic unicellular protists have lost the capacity to generate ATP via oxidative phosphorylation (with some exceptions 16,17 ). Instead, these eukaryotes generate ATP from fermentation through substrate-level phosphorylation, coupled to the formation of H 2 (ref. 18 ). This reaction takes place in the cytoplasm or-more commonly-in hydrogenosomes. Hydrogenosomes are specialized organelles, which have independently evolved from mitochondria in evolutionarily distant organisms 18,19 . Some ciliates that contain hydrogenosomes also contain endosymbiotic methanogenic archaea that scavenge the produced H 2 , which thereby increases the energy yield of the reaction 20 .
In contrast to strict anaerobes that exclusively ferment, some facultatively anaerobic eukaryotes can perform anaerobic respiration 21 . Nitrate and/or nitrite reduction has previously been demonstrated for some foraminifera and Gromiida [22][23][24] , fungi 25,26 and the ciliate Loxodes 27 . The underlying molecular mechanism of eukaryotic denitrification and energy conservation is largely unknown. Here we report the discovery of an obligate endosymbiont of an anaerobic ciliate that provides its host with energy derived from denitrification.

Article
We observed a high abundance of motile unicellular eukaryotes throughout the anoxic layer (  Table 1). The ciliates exhibited negative aerotaxis, which indicates an obligate anaerobic lifestyle (Extended Data Fig. 3). In some cases, we observed fragmented and densely packed 4′,6-diamidino-2-phenylindole (DAPI) signals that might have depicted microbial prey in food vacuoles (Fig. 1, Extended Data Fig. 2). However, a substantial fraction of the ciliates observed in the anoxic hypolimnion also contained multiple intracellular DAPI signals that were reminiscent of endosymbionts. These putative endosymbionts did not show the typical cofactor F 420 autofluorescence that is indicative of methanogen-hydrogenosome assemblages (which are often found in anaerobic ciliates) nor did they hybridize with a general archaeal oligonucleotide probe (Extended Data Fig. 2). We further confirmed the absence of methanogenic archaea in the ciliate by single-ciliate transcriptome sequencing (Supplementary Table 13, Supplementary Discussion). Instead, we obtained clear fluorescent signals after hybridization with a general bacteria-specific oligonucleotide probe (EUB-I) (Supplementary Table 3), which identified the potential ciliate endosymbionts as bacteria (Extended Data Fig. 2).

Endosymbiont and host phylogeny
To identify the putative endosymbiont, we used DNA extracted from the anoxic lake water for metagenome sequencing (Supplementary Table 4). We identified a small (about 290 kb), circular metagenome-assembled genome with typical endosymbiotic features (as described in 'Bacterial genome with endosymbiotic features'). The 16S rRNA gene sequence retrieved from the endosymbiont circular metagenome-assembled genome shared the highest identities (about 86%) with Legionella clemsonensis and 'Candidatus Berkiella cookevillensis' C99, which is an intranuclear bacterium of Acanthamoeba polyphaga. Given the high degree of sequence divergence between the endosymbiont and related bacteria, we named this organism 'Candidatus Azoamicus ciliaticola' (see Methods for etymology).
Our phylogenetic analysis showed that 'Ca. A. ciliaticola' belongs to the understudied eub62A3 group of the Gammaproteobacteria (Fig. 1e,  Extended Data Fig. 4). The eub62A3 group (which we refer to as the 'Candidatus Azoamicus' group) probably represents its own order within Gammaproteobacteria. The 'Ca. Azoamicus' group appears to consist of at least two distinct subgroups. Sequences affiliated with subgroup A are closely related to 'Ca. A. ciliaticola' (98.5-99.1% sequence identity) and have been retrieved from geographically distant anoxic freshwater lakes and reservoirs (Fig. 1e). Subgroup B contains sequences that are more divergent from 'Ca. A. ciliaticola' (89.5-92.9% sequence identity) and that have been retrieved from more diverse aquatic habitats (Fig. 1e). We used a fluorescently labelled 'Ca. Azoamicus'-specific oligonucleotide probe (eub62A3_813) (Extended Data Fig. 5, Methods, Supplementary Table 3), and found that the endosymbionts were exclusively located inside small ovoid (25.4 ± 3.2-μm long, 19.5 ± 2.0-μm wide (±s.d. from average); n = 9 cells) ciliate hosts ( Fig. 1, Extended Data Fig. 2). In 2018, all the ciliates we found at 180 m depth contained the 'Ca. A. ciliaticola' symbiont (Supplementary Table 1). We extracted DNA from individually picked ciliates from the deepest depth (189 m, which was sampled in 2019) and amplified 'Ca. A. ciliaticola' 16S rRNA gene using specific primers (Supplementary Table 2). We successfully retrieved sequences that were closely related to 'Ca. A. ciliaticola' (96.5-99.9% identity) from the picked ciliates. Our analysis of the partial 18S rRNA gene amplified from the same DNA extracts using ciliate-specific primers 28 (Supplementary Fig. 1) identified all of the picked ciliates as members of the class Plagiopylea within the Ciliophora phylum (Fig. 1f). We further confirmed their Ciliophora affiliation by analyses of single-copy orthologous gene transcripts from single-ciliate transcriptomes (Extended Data Fig. 6). Plagiopyleans are anaerobic or micro-aerobic ciliates that are typically found in anoxic freshwater or marine habitats. They are generally thought to rely on hydrogenosomes, and some host methanogenic archaea 29 . The class Plagiopylea is phylogenetically divided into two clades 30 . One clade contains well-described members of the order Plagiopylida (such as Plagiopyla frontata and Trimyema compressum), and the second clade contains our 'Ca. A. ciliaticola'-associated plagiopylean ciliate as well as diverse 18S rRNA gene sequences from freshwater and marine anoxic habitats (Fig. 1f). Our phylogenetic analyses thus indicated that 'Ca. A. ciliaticola' represents an endosymbiont of an anaerobic freshwater plagiopylean ciliate.

Bacterial genome with endosymbiotic features
The closed genome of 'Ca. A. ciliaticola' is very small (292,520 bp), and has a notably low average G + C content (24.4%) and a high protein-coding density (94.0%) (Fig. 2a). It has retained 310 protein-coding genes, 1 ribosomal RNA operon (16S, 23S and 5S), 35 transfer RNAs that decode all 20 standard amino acids (genetic code 11) and 1 transfer-messenger RNA; as such, it is, to our knowledge, the smallest protist endosymbiont genome reported to date. Genomes of a similar size and with similar general features have thus far been found only in obligate endosymbionts of insects 31,32 (Fig. 2b). On the basis of these similarities, we concluded that 'Ca. A. ciliaticola' is an obligate endosymbiont.
'Candidatus A. ciliaticola' encodes a similar proportion of genes related to translation, replication and repair to that encoded by other highly reduced endosymbionts of ciliates and insects (Fig. 2c). The genome encodes several subunits of the DNA polymerase III holoenzyme (dnaE, dnaQ and dnaX), DNA-directed RNA polymerase (rpoA, rpoB, rpoC, rpoD, rpoH and rpoZ) and a complete gene set of 30S and 50S ribosomal proteins. These genes were highly transcribed and accounted for around 20% of cumulative transcripts per million (Extended Data Fig. 7). Therefore, 'Ca. A. ciliaticola'-despite its reduced genomeappears to be capable of self-replication inside its host.
By contrast, the 'Ca. A. ciliaticola' genome exhibits extensive loss of the genes that underlie cell wall, membrane and envelope biogenesis. The genetic repertoire for the biosynthesis of vitamins and amino acids is also markedly reduced, which is notably different from other endosymbionts of insects or ciliates (Fig. 2c, Extended Data Fig. 7). As many endosymbionts provide essential amino acids or vitamins to supplement the nutritionally poor diets of their hosts, they often retain extensive biosynthetic pathways for these compounds 32 . The 'Ca. A. ciliaticola' genome encodes only three copies of a tyrosine and tryptophan transporter (tyrP) and a glutamate and γ-aminobutyrate (glutamate/γ-aminobutyrate) antiporter that might serve to import aromatic amino acids and glutamate into the cell. Notably, 'Ca. A. ciliaticola' appears to depend on its host for most of the other essential cellular building blocks (for example, nucleotides, phospholipids and lipopolysaccharides), as no genes for their biosynthesis are encoded in the genome. It is therefore puzzling  Table 9) between 'Ca. A. ciliaticola' (magenta), J. libera mitochondrion (blue), selected insect and ciliate endosymbionts (orange), and Legionella clemsonensis (grey) as an example of a related free-living bacterium. Numbers are as described in b; 6 denotes L. clemsonensis. The asterisk highlights the presence of an anaerobic respiratory chain. Single-letter codes of COG functional categories are shown in parentheses.

Article
that, similar to other obligate endosymbionts 33 , the 'Ca. A. ciliaticola' genome largely lacks transport systems for these metabolites (Supplementary Tables 5, 6, Supplementary Discussion). Other genes that have previously been shown to be related to nutritional (for example, those that encode pectinases 34 ) or defensive symbioses known from ciliates 35 are also absent from the genome.

Anaerobic respiration and energy metabolism
In contrast to most other functional gene categories, genes related to respiration and energy production and conversion (Figs. 2c, 3a, Supplementary Table 5) are abundantly retained in the 'Ca. A. ciliaticola' genome. 'Candidatus A. ciliaticola' encodes a complete ATP-generating electron transport chain, including NADH dehydrogenase (nuoA to nuoN), cytochrome bc 1 complex (qcrA, qcrB and qcrC) and ATP synthase (atpA to atpH). In many endosymbionts, genes related to respiration and energy production and conversion are affected by marked losses; some endosymbionts are even thought to be ATP parasites 32 . Instead, a substantial fraction of the 'Ca. A. ciliaticola' genome is dedicated to energy production and conversion, which is a feature that it shares with all known mitochondrial genomes (Fig. 2c). The 'Ca. A. ciliaticola' genome does not encode genes for aerobic respiration, such as terminal oxidases (for example, cytochrome c oxidase and cytochrome bd oxidase). In principle, the terminal oxidase genes could have been relocated to the nuclear genome (as is the case with many endosymbiont genes). However, electron transport chain complexes I, II, IV and V are conserved in the mitochondrial core genome across many eukaryotes, and it is expected that genes that encode components of the electron transport chain are amongst the least likely to be transferred to the nucleus 11 . We also did not find transcripts for mitochondrially encoded cytochrome c oxidase in the host transcriptome (Supplementary Table 10, Supplementary Discussion). This confirms the obligate anaerobic nature of the host, as was already indicated by its negative aerotactic behaviour (Extended Data Fig. 3).
The 'Ca. A. ciliaticola' genome encodes a complete gene set for respiratory denitrification (Fig. 3a, Supplementary Table 5). The gene set contains all four core enzymes: nitrate reductase (narG, narH and narI), copper-containing nitrite reductase (nirK), nitric oxide reductase (norB and norC) and nitrous oxide reductase (nosZ). These genes are complemented by a wide variety of accessory proteins, such as cytochromes c 4 and c 5 for redox coupling, enzyme maturation factors (narJ, nosR, nosD, nosF, nosY and nosL), and nitrate and nitrite transporters (narK and narT). 'Candidatus A. ciliaticola' encodes a molybdate import system (modA, modB and modC) and a pathway for the biosynthesis of bis-molybdopterin guanine dinucleotide, which is an essential cofactor of nitrate reductase. In eukaryotes, this pathway is-for the most part-a cytosolic process that is encoded by nuclear genes 36 . Similarly, a pathway of iron-sulfur-cluster biosynthesis (Suf-type) (sufB, sufC, sufD and sufS) is also encoded in the 'Ca. A. ciliaticola' genome; this was the only pathway that we detected for iron-sulfur-cluster biosynthesis   There is a highly consistent transcriptome profile between years and an exceptionally high transcription of genes related to energy conservation, ATP production and transport, chaperones (groE, groS and groL) and denitrification (orange, red, purple and blue shading, respectively). Coverage was corrected for sequencing depth; the higher coverage in the 2018 sample is due to the higher endosymbiont abundance in that year. Genes related to denitrification (blue), electron transport chain (orange), tricarboxylic acid cycle (grey) and ATP generation as well as transport (red) are highlighted. The highlighted genes account for more than 55% of all transcripts. Further information is provided in Supplementary Table 5). c, Correlation of ciliate abundance and denitrification rates ( 30 N 2 production) in water from the anoxic hypolimnion (189 m, May 2019) with and without ciliates. Each data point represents a volumetric denitrification rate calculated from the linear regression of six individual time points of an experiment. Error bars represent s.e. from linear regression. Denitrification rates in incubations without ciliates are due to the activity of denitrifying bacteria that are still present in the water.
in the ciliate host. We did not detect transcripts that pertain to the Isc-type pathway of iron-sulfur-cluster biosynthesis (which operates in hydrogenosomes, mitochondria and mitochondria-related organelles) in the single-ciliate transcriptome (Supplementary Table 11).
Although the trait of respiratory denitrification is widespread among free-living prokaryotes 37,38 , to our knowledge no obligate endosymbiont described to date encodes a denitrification pathway. We confirmed active denitrification by ciliates using 15 N stable isotope incubations (Fig. 3c, Extended Data Fig. 8, Supplementary Discussion). Possible electron donors for denitrification might be di-and/or tri-carboxylates provided by the tricarboxylic acid cycle, such as succinate and malate (Supplementary Discussion). Malate is used by many known energy-producing anaerobic mitochondria or hydrogenosomes of ciliates or other protists 4 . The high transcription of malate dehydrogenase (mdh) and a putative 2-oxoglutarate and malate (2-oxoglutarate/malate) transporter (yflS) (Fig. 3b) indicates that malate also has a key role in the metabolism of 'Ca. A. ciliaticola'. Similarly, genes related to energy conservation-including ATP synthase (atpA to atpH) and denitrification genes (narG, narH, narI, nirK, norB, norC and nosZ)-were among those showing the highest transcription ( Fig. 3b) and, altogether, we found that more than half of the transcriptional effort of 'Ca. A. ciliaticola' was devoted to energy production and conversion (Extended Data Fig. 7).
Crucially, the 'Ca. A. ciliaticola' genome also contains a putative ATP and ADP (hereafter, ATP/ADP) translocase gene (tlcA) that encodes a single-domain nucleotide transport protein. These nucleotide transport proteins have canonical roles in energy transfer in plastids and intracellular parasites, although these might not be their only functions 39 . The 'Ca. A. ciliaticola' nucleotide transport protein has several conserved residues that are critical for function and substrate specificity of ATP/ ADP translocases (Extended Data Fig. 9, Supplementary Discussion). Moreover, the tlcA gene was among the ten highest transcribed 'Ca. A. ciliaticola' genes, together with other genes that are involved in energy conservation (Fig. 3b). On this basis, we speculate that the TlcA nucleotide transporter serves to exchange ATP and ADP between 'Ca. A. ciliaticola' and its host. As 'Ca. A. ciliaticola' lacks the ability to synthesize any nucleotides de novo, this nucleotide transport protein might also be involved in the uptake of nucleotides other than ATP and ADP.
As is known from other obligate endosymbionts, the genome of 'Ca. A. ciliaticola' has presumably preserved traits that are beneficial to the host. Given the fact that the highly reduced genome of 'Ca. A. ciliaticola' has retained an extensive genetic potential for energy metabolism, the main function of this endosymbiont thus appears to be the production of ATP for its host. In this respect, the function of 'Ca. A. ciliaticola' is strongly evocative of mitochondria and hydrogenosomes (that is, eukaryotic energy-providing organelles). On the basis of our combined metagenomic and transcriptomic results, we propose that 'Ca. A. ciliaticola' functions as a respiratory endosymbiont that conserves and provides energy from anaerobic respiration for its plagiopylean host.

Evolution of the respiratory symbiosis
Unicellular eukaryotes that have secondarily adapted to an anaerobic lifestyle often possess mitochondria-related organelles such as hydrogenosomes. These organelles have retained their energy-generating function-although they have often lost their genomes, and their enzymes are predominantly nuclear-encoded and expressed by the eukaryotic host 40 . When analysing the single-cell transcriptomes of the ciliate host, we found transcripts that potentially encode hydrogenosomal proteins (Supplementary Tables 11, 12, Supplementary Methods, Supplementary Discussion). This suggests that our ciliate host-as with all eukaryotes known to date-may contain remnants of a mitochondrial organelle, which is probably capable of hydrogenosomal metabolism (Fig. 4).
The putative presence of hydrogenosomes in the extant host implies that, at some point, its ancestor possessed aerobic mitochondria. However, in a host that is capable of aerobic respiration, the acquisition of a denitrifying endosymbiont is unlikely to have increased the fitness of the host sufficiently to warrant stable integration. It is thus more likely that the ciliate host was already a hydrogenosome-containing obligate anaerobe before the acquisition of the predecessor of 'Ca. A. ciliaticola' (Fig. 4). A fermenting eukaryote that possessed only rudimentary means of energy generation would have profited considerably from the acquisition of a bacterium with the capacity for anaerobic nitrate respiration and chemiosmotic ATP production. The positive effect of the 'Ca. A. ciliaticola' endosymbiont on the fitness of the ciliate host could explain our observation that only 'Ca. A. ciliaticola'-containing ciliates were observed in the deep anoxic nitrate-containing waters of Lake Zug (Supplementary Table 1).
The predecessor of 'Ca. A. ciliaticola' may have entered its host as a parasite, as many members of the related Legionellales and Francisellales groups are parasitic. Alternatively, the bacterial predecessor could have been acquired through endocytosis and then persisted intracellularly after managing to escape digestion, as has previously been suggested for some bacteria 41 . In either case, the protein that was probably fundamental for the integration of the bacterium into its host is the ATP/ADP transporter. Phylogenetic analyses showed that the putative ATP/ADP translocase of 'Ca. A. ciliaticola' clusters with mostly alphaproteobacterial nucleotide transporter sequences from uncharacterized metagenomic sequences related to Holosporales or Caedibacter (Extended Data Fig. 10), which are known to be intracellular parasites or endosymbionts  Article of ciliates 42,43 . Considering that the free-living predecessor of 'Ca. A. ciliaticola' was a gammaproteobacterium, we speculate that the ATP/ADP translocase might have been obtained via lateral gene transfer from an alphaproteobacterial donor (for example, Holospora-related) (Supplementary Discussion). At this point, it is not clear whether the lateral gene transfer happened before or after the engulfment of the predecessor of 'Ca. A. ciliaticola' by the ciliate (Fig. 4).
In its extant form, 'Ca. A. ciliaticola' encodes only the denitrification respiratory chain. This is noteworthy as almost all pro-and eukaryotic denitrifying organisms known to date possess a hybrid respiratory chain that is also capable of oxygen respiration 38,44 . On this basis, it seems likely that the predecessor of 'Ca. A. ciliaticola' acquired by the ciliate host was a facultatively anaerobic denitrifying bacterium (Fig. 4). Subsequently, it might have lost its capacity for oxygen respiration when it became superfluous in a ciliate that exclusively inhabited anoxic waters. However, any gene loss in 'Ca. A. ciliaticola' might also be the result of genetic drift, which is known to affect obligate endosymbionts with small population sizes and restricted gene flow 31 .
Over the course of its existence, 'Ca. A. ciliaticola' has evolved into an obligate endosymbiont with a distinct role in respiration and energy generation for its host; in eukaryotes, these roles are traditionally reserved for mitochondria and related organelles. Even though the ciliate host might contain mitochondria-related organelles (that is, putative hydrogenosomes), it appears that quintessential mitochondrial functions related to respiration, the electron transport chain, oxidative phosphorylation and iron-sulfur-cluster biosynthesis are taking place in its endosymbiont.
'Candidatus A. ciliaticola' therefore represents an example of an obligate endosymbiont that has retained cellular functions that are markedly similar to those of mitochondria, although it did not originate from the mitochondrial line of descent. This discovery provokes the question of whether other prokaryotic endosymbionts may enable eukaryotes to tap into the broad pool of electron acceptors that is available in nature.

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Methods
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.

Etymology
The designation 'Azoamicus' combines the prefix azo-(New Latin, pertaining to nitrogen) with amicus (Latin, masculine noun, friend); thus giving azoamicus ('friend that pertains to nitrogen'), alluding to its role as denitrifying endosymbiont. 'Ciliaticola' combines ciliate (referring to a group of ciliated protozoa) with the suffix -cola (derived from the Latin masculine noun incola, dweller, inhabitant), thus meaning 'dwelling within a ciliate'.

Geochemical profiling
We carried out sampling for geochemical profiling in September 2016 and October 2018 at a single station located in the deep, southern lake basin of Lake Zug (about 197-m water depth) (47° 06′ 00.8′′ N, 8° 29′ 35.0′′ E). In September 2016, we used a multi-parameter probe to measure conductivity, turbidity, depth (pressure), temperature and pH (XRX 620, RBR). Dissolved oxygen was monitored online with normal and trace micro-optodes (types PSt1 and TOS7, Presens) with detection limits of 125 and 20 nM, respectively, and a response time of 7 s. In October 2018, we used a CTD (CTD60, Sea&Sun Technology) equipped with a Clark-type oxygen sensor (accuracy ± 3%, resolution 0.1%) to record oxygen profiles.

Sample collection
Water for bulk DNA and RNA analyses was collected in September 2016 and October 2018. Sample collection for DNA and RNA extraction in September 2016 has previously been described 46 . In October 2018, water was sampled with a Niskin bottle (Hydro-Bios) from 160 m, 170 m and 180 m. For each depth, 2 l of lake water was directly filtered on board our boat onto 0.22-μm Sterivex filter cartridges (Merck Millipore) using a peristaltic pump, subsequently purged with RNAlater preservation solution (Life Technologies) and stored at −20 °C until further processing. For fluorescence in situ hybridization (FISH) analyses, water from the same depths was fixed on board the boat with formaldehyde (1.5% final concentration; Electron Microscopy Sciences) and incubated in a chilled cool box for about 6 h before filtration onto 3-μm polycarbonate filters (Merck Millipore). Additional FISH samples using the same approach were collected in May 2019 from 189 m water depth.
Water for incubation experiments and single-ciliate PCR was sampled in May 2019 from 189-m water depth using a 10 l Go-Flo bottle (General Oceanics), filled into 2.5-l glass bottles without headspace, closed with butyl rubber stoppers and kept cold (at about 4 °C) and dark until further handling. During sampling, oxygen contamination was minimized by overflowing the bottle with anoxic lake water.
For combined FISH and differential interference contrast microscopy analyses, individual live ciliates were picked from lake water (from 186-m depth, collected February 2020) and directly fixed on microscope slides. In brief, microscope slides were treated with 0.1 mg ml −1 poly-l-lysine for 10 min at room temperature, washed with MilliQ water and dried. Ciliates were pre-enriched by gravity flow of bulk lake water through a 5-μm membrane filter and picked using a glass capillary under a binocular microscope. Picked ciliates were transferred into a droplet of formaldehyde (2% in 0.1× PBS, pH 7.6) on poly-l-lysine-coated microscope slides, incubated (for 1 h at room temperature) and washed with MilliQ water. FISH was performed as described in 'Double-labelled oligonucleotide probe fluorescence in situ hybridization and microscopy'.

Nutrient measurements
Water samples for measurements of nutrients (ammonium, NO x and nitrite) were retrieved with a syringe sampler from 15 discrete depths at and below the base of the oxycline. Forty ml of water was directly injected into a 50-ml Falcon tube containing 10 ml of OPA reagent for fluorometric ammonium quantification 47 . In 2018, ammonium concentration was determined using the same method, except that the lake water was immediately sterile-filtered after sampling and frozen at −20 °C until further processing. For NO x quantification, 10 ml of water was sterile-filtered into a 15-ml Falcon tube and combined nitrate and nitrite concentration was determined by a commercial QuAAtro Segmented Flow Analyzer (SEAL Analytical).
Clone library construction and Sanger sequencing 'Candidatus A. ciliaticola'-specific 16S rRNA gene primers were designed on the basis of the 'Ca. A. ciliaticola' circular metagenome-assembled genome sequence. Primers targeted the intergenic spacer regions about 50 bp up-and downstream of the 16S rRNA gene, resulting in a 1,568-bp-long PCR product. For clone library construction, the 'Ca. A. ciliaticola' 16S rRNA gene was amplified by a nested PCR approach from the same DNA extract used for metagenome sequencing obtained in September 2016 from 160-m water depth using the newly designed 'Ca. A. ciliaticola'-specific primers (eub62A3_29F and eub62A3_1547R) followed by PCR amplification with general bacterial 16S rRNA gene primers (8F and 1492R) (Supplementary Table 2). Cloning and construction of the clone library is described in more detail in Supplementary Methods. Inserts of purified plasmids from five clones were sequenced by Sanger sequencing using the BigDye Terminator v.3.1 sequencing kit (Thermo Fisher Scientific) and primers M13f or M13r. The sequencing PCR contained 3 μl purified plasmid, 0.5 μl 10× sequencing buffer, 0.5 μl primer (10 μM) and 1 μl BigDye reagent. The PCR reactions were performed as follows: 99 cycles (1 °C s −1 ramp) of denaturation (10 s at 96 °C), annealing (5 s at 60 °C) and elongation (4 min at 60 °C). The PCR products were purified using gel filtration (Sephadex G-50 Superfine, Amersham Bioscience) followed by Sanger sequencing (3130xl genetic analyser, Applied Biosystems). The Sanger sequences were quality-trimmed and assembled using Sequencher v.5.4.6 and standard settings before trimming vector and primer sequences.

Probe design for fluorescence in situ hybridization
To visualize 'Ca. A. ciliaticola' cells in the environment, we designed a specific FISH probe on the basis of the 'Ca. A. ciliaticola' circular metagenome-assembled genome 16S rRNA gene sequence and closely related sequences within the clade eub62A3. The 'Ca. A. ciliaticola' 16S rRNA gene sequence was imported into Arb 48 v.6.1 and aligned to the SILVA SSU Ref NR 99 132 database using the SINA-Aligner 49 . A FISH probe specific for 'Ca. A. ciliaticola' and most members of clade eub62A3 was designed (probe eub62A3_813 5′ CTAACAGCAAGTTTTCATCGTTTA 3′) (Supplementary Table 3) using the probe design tool implemented in Arb, and further manually refined and evaluated in silico using MathFISH 50 . The newly designed probe eub62A3_813 targets 'Ca. A. ciliaticola' and 78% of the 'Ca. Azoamicus' subgroup A and B sequences included in SILVA SSU Ref NR 99 138 (7 out of 9; the 2 sequences that are not targeted belong to 'Ca. Azoamicus' subgroup B), and shows no nontarget hits. Some sequences in the database had only 1 or 2 weak mismatches (<0.5 weighted mismatches) to the probe sequence. To ensure specificity in our FISH experiments and exclude the detection of nontarget organisms, we designed unlabelled competitor probes for these sequences with weak mismatches to the probe (competitor 1, 5′ CTA ACAGCAAGTTCTCATCGTTTA 3′ and competitor 2, 5′ CCAACAGCAAGTT CTCATCGTTTA 3′) (Supplementary Table 3). These competitor probes were included in all FISH experiments (excluding clone-FISH). To further ensure that no nontarget organisms were present in our samples, we searched for 16S rRNA gene reads with perfect matches against the eub62A3_813 probe sequence in one metagenome (Zug 2018, 180 m). Almost all of the reads (82 out of 86) had >98% identity with the 16S rRNA gene sequence of 'Ca. A. ciliaticola'. The remaining reads shared >94% identity with 'Ca. A. ciliaticola' and all shared as top hits sequences Article belonging to 'Ca. Azoamicus' subgroup A when blasted against the NCBI nr database (accessed June 2020).

Clone-FISH
Clone-FISH was performed as previously described 51 . In brief, a purified plasmid containing the 'Ca. A. ciliaticola' 16S rRNA gene sequence (as described in 'Clone library construction and Sanger sequencing') in the correct orientation (confirmed by PCR using M13F and 1492R primers) was transformed into electrocompetent Escherichia coli JM109(DE3) cells (Promega) by electroporation using the Cell Porator and Voltage Booster System (Gibco) with settings 350 V, 330 μF capacitance, low ohm impedance, fast charge rate and 4 kΩ resistance (Voltage Booster). After electroporation, cells were transferred into SOC medium (Sigma Aldrich), incubated for 1 h at 37 °C and plated onto LB plates containing 100 mg l −1 kanamycin. After incubation overnight at 37 °C, 4 clones were picked and the presence of the insert was checked with PCR (primers M13F and 1492R) as described in 'Clone library construction and Sanger sequencing', followed by gel electrophoresis. An insert-positive clone was selected at random and grown in LB medium containing 100 mg l −1 kanamycin at 37 °C until optical density at 600 nm reached 0.37. Transcription of the plasmid insert was induced using isopropyl β-d-1-thiogalactopyranoside (IPTG) (1 mM final concentration). After addition of IPTG, cells were incubated for 1 h at 37 °C followed by addition of 170 mg l −1 chloramphenicol and subsequent incubation for 4 h. Cells were collected by centrifugation, fixed in 2% formaldehyde solution for 1 h at room temperature, washed and stored at 4 °C in phosphate-buffered saline (pH 7.4) containing 50% ethanol until further processing. Formamide melting curves 52 were carried out using a 'Ca. Azoamicus'-specific, HRP-labelled probe (eub62A3_813) (Extended Data Fig. 5). In brief, cells were applied to glass slides. Permeabilization with lysozyme, peroxidase inactivation, hybridization (10%, 30%, 35%, 40%, 45% and 50% formamide) and tyramide signal amplification (Oregon Green 488) was performed as previously described 53 . For each formamide concentration, images of three fields of view were recorded using the same exposure time for all formamide concentrations, which was optimized at 10% formamide all same settings using an Axio Imager 2 microscope (Zeiss) and analysed using Daime 2.1 54 .

Double-labelled oligonucleotide probe fluorescence in situ hybridization and microscopy
Hybridization with double-labelled oligonucleotide probes (terminally double-labelled with Atto488 dye; details of the probe are in Supplementary Table 3) (Biomers) and counterstaining with DAPI was performed as previously described 55 . In brief, samples (either cut filter sections or ciliates picked and fixed on a glass slide, as described in 'Sample collection') were incubated in hybridization buffer containing 25% formamide and 5 ng DNA μl −1 probe (the same concentration was used for eub62A3_813 competitor 1 and 2) for 3 h at 46 °C, and subsequently washed in prewarmed washing buffer (5 mM EDTA, 159 mM NaCl) for 30 min at 48 °C. After a brief MilliQ water wash, samples were incubated for 5 min at room temperature in DAPI solution (1 μg ml −1 ), briefly washed in MilliQ water and air-dried. Filter sections were mounted onto glass slides. Samples were embedded in Prolong Gold Antifade Mountant (Thermo Fisher Scientific), and left to cure for 24 h. Confocal laser scanning and differential interference contrast microscopy were performed on a Zeiss LSM 780 (Zeiss, 63× oil objective, 1.4 numerical aperture, with differential interference contrast prism). Z-stack images were obtained to capture entire ciliate cells and fluorescence images of FISH probe and DAPI signals were projected into 2D for visualization. Cell counting was performed using a Axio Imager 2 microscope (Zeiss) in randomly selected fields of view (40× objective, grid length = 312.5 μm) on polycarbonate filters (3-μm pore size, 32-mm effective filter diameter; Merck Millipore) onto which 0.5 l PFA-fixed lake water was filtered.
For light microscopy, live ciliates were picked from Lake Zug water (May 2019, 189 m) and prepared for live ciliate imaging using light microscopy as previously described 56 on an Axio Imager 2 microscope (Zeiss). For image acquisition and processing, Zeiss ZEN (blue edition) 2.3 was used.

Scanning electron microscopy
Ciliates sampled in February 2020 were individually picked under a binocular microscope, and washed in droplets of sterile-filtered lake water. Washed ciliates were then transferred into approximately 200 μl fixative on top of a polylysine-coated silicon wafer (0.1 mg ml −1 poly-l-lysine for 10 min at room temperature) and fixed for 1 h at room temperature. The fixative contained 2.5% glutaraldehyde (v/v, electron microscopy grade) in PHEM buffer 57 (pH 7.4). Fixed ciliates attached to the silicon wafer were dehydrated in an ethanol series (30%, 50%, 70%, 80%, 96% and 100%) before automated critical-point drying (EM CPD300, Leica). Scanning electron microscopy was performed on a Quanta FEG 250 (FEI). Images were obtained using FEI xTM v.6.3.6.3123 at an acceleration voltage of 2 kV under high vacuum conditions and were captured using an Everhart-Thornley secondary electron detector. The image represents an integrated and drift corrected array of 128 images captured with a dwell time of 50 ns.

Single-ciliate PCR
Ciliates were picked from Lake Zug water (189 m, 2019) under the binocular microscope with a glass micropipette, and subsequently washed twice in drops of sterile nuclease-free water (Ambion) before being transferred into lysis buffer. DNA was extracted using Master-Pure DNA purification kit (Ambion) following the manufacturer's instructions with a final elution in 1× TE buffer (25 μl). Overall, DNA was extracted from four individual ciliates (S1-S4), five (C5) and ten pooled ciliates (C10) as well as no ciliate (control). 16S ('Ca. A. ciliaticola') and 18S rRNA gene sequences (ciliate host) were then separately amplified by PCR using primer pairs eub62A3_29F/_1547R and cil_384F/_1147R. PCR reactions (20 μl) with 'Ca. A. ciliaticola'-specific primers (eub62A3_29F/_1547R) were performed as described in 'Clone library construction and Sanger sequencing' with following modifications: 5 μl template, 58 °C annealing temperature and 40 amplification cycles. PCR with ciliate-specific primers (cil_384F/_1147R) was performed analogously with following modifications: 55 °C annealing temperature, 50 s elongation and 35 amplification cycles. The PCR reactions with primer pairs eub62A3_29F/_1547R were further amplified in a second round of PCR (under the same conditions) using 2 μl PCR reaction from the first round. For each PCR step, successful amplification of products was checked using gel electrophoresis as described in Supplementary Methods. PCR reactions were subsequently purified using QIAquick PCR purification Kit (Qiagen) according to the manufacturer's instructions with a final elution in sterile nuclease-free water (25 μl) (Ambion). Purified PCR reactions were subsequently sequenced using Sanger sequencing and processed as described in 'Clone library construction and Sanger sequencing' with the following modifications: primers eub62A3_29F, eub62A3_1547R (annealing temperature 58 °C) or cil_384F, cil_1147R (annealing temperature 55 °C). Two of the single-ciliate DNA extracts amplified with the endosymbiont-specific primers either showed a faint (S4) or no (S2) band and were not sequenced.

Nucleic acid extraction from lake water
Bulk DNA and RNA extraction as well as metagenome and metatranscriptome sequencing of lake water samples collected in 2016 have previously been described 46 . For samples from 2018, filters were purged of RNAlater, briefly rinsed with nuclease-free water and removed from the Sterivex cartridge. RNA and DNA was then extracted from separate filters using PowerWater RNA or DNA isolation kits (MoBio Laboratories) according to the manufacturer's instructions.

Metagenome and bulk metatranscriptome sequencing
For metagenomic sequencing, DNA libraries were prepared as recommended by the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). Sequencing-by-synthesis was performed on the Illumina HiSeq2500 sequencer (Illumina Inc.) with the 2 × 250-bp read mode. For metatranscriptomic sequencing, rRNA was removed (Ribo-Zero rRNA Removal Kit for bacteria (Illumina)) and an RNA-sequencing library was prepared according to the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing-by-synthesis was performed on the Illumina HiSeq3000 sequencer (Illumina) with the 1 × 150-bp read mode. Library preparation and sequencing was performed by the Max Planck Genome Centre Cologne (http://mpgc.mpipz.mpg.de/home/). Detailed information for each metagenomic and metatranscriptomic dataset can be found in Supplementary Table 4.

Genome assembly and finishing
The genome of 'Ca. A. ciliaticola' was reconstructed from a metagenomic dataset sampled in 2018, as follows: metagenomic reads (MG_18_C) were trimmed using Trimmomatic 58 v.0.32 as previously described 46 and assembled using metaSPAdes 59

Genome annotation and comparative analyses
Genome annotation was performed using a modified version of Prokka 62 v.1.13.3 to allow annotation of genes that overlap with tRNA genes. The annotation of key metabolic genes was manually inspected and refined using searches against NCBI non-redundant protein or conserved domain database 63 . Transmembrane transporters were predicted and classified using the Transporter Automatic Annotation Pipeline web service 64 and the Transporter Classification Database 65 . Pseudogenes were predicted using pseudo finder 66 v.0.11 and standard settings. Circular genome maps were created using DNAplotter 67 v.18.1.0.
For comparative analyses, protein-coding CDS encoded in the genomes of insect endosymbionts (C. ruddii PV, AP009180.1 and B. aphidicola BCc, CP000263.1), mitochondrion of J. libera (NC_021127) and a free-living relative of 'Ca. A. ciliaticola' (L. clemsonensis, CP016397.1) were downloaded from NCBI GenBank. Additionally, protein-coding CDS of the ciliate endosymbiont C. taeniospiralis (PGGB00000000.1) were obtained using Prokka annotation. Classification of functional categories was performed using the eggNOG-mapper v.1 web service 68 with mapping mode DIAMOND and standard settings. The classification of the functional category C (energy production and conversion) for 'Ca. A. ciliaticola' was modified to also include norB and nirK, which were grouped by eggNOG into different categories (Q and P, respectively).

Metatranscriptomic analyses of bulk water samples
Raw metatranscriptomic Illumina reads trimming and removal of rRNA sequences was performed as previously described 46 . Non-rRNA reads were then mapped to the genome of 'Ca. A. ciliaticola' using Bowtie2 71 v.2.2.1.0 and standard parameters. Sorted and indexed BAM files were generated using samtools 72 v.0.1.19 and transcripts per feature (based on the Prokka annotation) were quantified using EDGE-pro 73

Phylogenetic analyses
The full-length 16S rRNA gene sequence was retrieved from the circular metagenome-assembled genome of 'Ca. A. ciliaticola' using RNAmmer 77 v.1.5, aligned using the SILVA incremental aligner 49 (SINA 1.2.11) and imported to the SILVA SSU NR99 database 45 (release 132) using ARB 48 v.6.1. Additional closely related 16S rRNA gene sequences were identified by BLASTN in the NCBI non-redundant nucleotide database and JGI IMG/M 16S rRNA Public Assembled Metagenomes (retrieved July 2018) and also imported into ARB. A maximum-likelihood phylogenetic tree of 16S rRNA gene sequences was calculated using RAxML 78 v.8.2.8 integrated in ARB with the GAMMA model of rate heterogeneity and the GTR substitution model with 100 bootstraps. The alignment was not constrained by a weighting mask or filter. For the complete tree shown in Extended Data Fig. 4, additional 'Ca. A. ciliaticola' sequences obtained from a clone library and individual single ciliates were added to the tree using the Parsimony 'Quick add' algorithm implemented in ARB.
For the ciliate phylogeny, sequences obtained from Sanger sequencing of picked ciliates were added to the EukRef-Ciliphora 30 Plagiopylea subgroup alignment using MAFFT 79 online service version 7 (argument:--addfragments). An additional metagenome-assembled full-length 18S rRNA gene sequence assigned to Plagiopylea was obtained using phyloFlash 80 v.3.0 from one of the Lake Zug metagenomes (MG_18_C) and also added to the alignment (argument:--add). A phylogenetic tree was calculated using IQ-TREE webserver (http://iqtree.cibiv.univie. ac.at) running IQ-TREE 81 1.6.11 with default settings and automatic substitution model selection (best-fit model: TIM2+F+I+G4). Phylogenetic trees were visualized using the Interactive Tree of Life v.4 web service 82 .
For the ATP/ADP translocase phylogenetic tree, amino acid sequences were retrieved from NCBI RefSeq (250 top hits) and NCBI nr (15 top hits) (both accessed June 2019) using NCBI blastp web-service 83 with the amino acid sequence of ATP/ADP translocase sequence of 'Ca. A. ciliaticola' (ESZ_00147) as query. Additional amino acid sequences of characterized nucleotide transporters listed in Supplementary Table 8 were also added. After removal of duplicates, sequences were clustered at 95% identity using usearch 84 v.8.0.1623 and aligned using MUSCLE 69 v.3.8.31. Phylogenetic tree construction using IQ-TREE (best-fit model: LG+F+I+G4) and visualization was performed as described for the 18S rRNA gene phylogenetic tree.

Incubation experiments
Incubation experiments were performed to provide experimental evidence for the denitrifying activity linked to the ciliate host. Three incubations were set up that contained (a) no ciliates (filtered fraction), (b) lake water that was enriched in ciliates (enriched fraction) and (c) bulk lake water. For these experiments, lake water was size-fractionated using a 10-μm polycarbonate filter (Merck Millipore) under N 2 atmosphere in a glove bag at 12 °C. Enriched and filtered fractions were obtained by gravity filtration of 0.5 l water until 0.25 l supernatant was left. Thus, in the enriched fraction, ciliates from 0.5 l lake water were concentrated in 0.25 l lake water. In the filtered fraction, organisms larger than 10 μm (including ciliates) were filtered out. Both the enriched water (plus filter) and the filtered water were transferred to separate serum bottles (no headspace) and closed with butyl rubber stoppers. For bulk incubations, unfiltered water was directly filled into 250 ml serum bottles under N 2 atmosphere.
Denitrification potential was assessed by measuring the production of 30 N 2 over time in 15 N-nitrite and 15 N-nitrate amended incubations by isotope ratio mass spectrometry (Isoprime Precision running ionOS v.4.04, Elementar). A 30 ml helium headspace was set for the 250 ml serum bottles and the water was degassed with helium for 10 min to ensure anoxic conditions and reduce N 2 background. A 15 N-labelled mixture of nitrate and nitrite (20 μM and 5 μM final concentration, respectively; Sigma Aldrich) was supplied at a 99% labelling percentage and the water was incubated for a total of 30 h at 4 °C in the dark. Subsamples of the headspace were taken at regular time intervals during the incubation by withdrawing 3 ml of the gaseous headspace and simultaneously replacing the removed volume by helium. This gas sample was transferred into 12 ml Exetainers (LabCo) that were pre-filled with helium-degassed water and stored until analysis. 30 N 2 in the gas samples was measured on an isotope ratio mass spectrometer, and denitrification rates were calculated from the slope of the linear increase of 30 N 2 in the headspace over the time course of the experiments. The rate of 30 N 2 production was corrected for dilution of the headspace introduced by subsampling and by the measured total 15 N labelling percentage. 'Ca. A. ciliaticola'-containing ciliate abundance in the different incubation bottles was assessed by microscopic counts after cell fixation, FISH hybridization (eub62A3_813) and DAPI staining as described in 'Double-labelled oligonucleotide probe fluorescence in situ hybridization and microscopy'.

Statistics and reproducibility
No statistical methods were used to predetermine sample size and experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
In Fig. 1a, the scanning electron microscopy image is a representative of n = 6 recorded images that were obtained from 1 experiment. In Fig 1b, the differential interference contrast image is a representative of n = 6 recorded images that were obtained from 1 experiment.
In Fig. 2c, Extended Data Fig. 2i. FISH fluorescence images (eub62A3_813 probe) are representative of n = 33 recorded images that were obtained from 5 independent experiments of 3 biological replicate samples.
In Fig. 2c, Extended Data Fig. 2f, h, j. DAPI fluorescence images are representative of n = 21 recorded images that were obtained from 5 independent experiments of 3 biological replicate samples.
In Extended Data Fig. 2a, the FISH fluorescence image (Arch915 probe) is representative of n = 6 images that were obtained from 1 experiment. In Extended Data Fig. 2b, d, F 420 autofluorescence images are representative of n = 11 recorded images that were obtained from 3 independent experiments of 1 sample. In Extended Data Fig. 2c, g, FISH fluorescence images (EUB-I probe) are representative of n = 15 images obtained from 3 independent experiments of 2 biological replicate samples. In Extended Data Fig. 2e, the FISH fluorescence image (NON338 probe) is representative of n = 15 recorded images that were obtained from 3 independent experiments of 2 biological replicate samples.
In Extended Data Fig. 5a, each of the 6 FISH fluorescence images (eub62A3_813 probe) is representative of n = 3 images from 1 experiment.
For the fluorescence images shown, the number of analysed cells was typically much higher (n > 100) than the ones that were eventually recorded.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.