Single-cell Microbiomics Unveils Distribution and Patterns of Microbial Symbioses in the Natural Environment.

Protist-bacteria associations are extremely common. Among them, those involving ciliates of the genus Euplotes are emerging as models for symbioses between prokaryotes and eukaryotes, and a great deal of information is available from cultured representatives of this system. Even so, as for most known microbial symbioses, data on natural populations is lacking, and their ecology remains largely unexplored; how well lab cultures represent actual diversity is untested. Here, we describe a survey on natural populations of Euplotes based on a single-cell microbiomic approach, focusing on taxa that include known endosymbionts of this ciliate. The results reveal an unexpected variability in symbiotic communities, with individual hosts of the same population harboring different sets of bacterial endosymbionts. Co-occurring Euplotes individuals of the same population can even have different essential symbionts, Polynucleobacter and "Candidatus Protistobacter," which might suggest that replacement events could be more frequent in nature than previously hypothesized. Accessory symbionts are even more variable: some showed a strong affinity for one host species, some for a sampling site, and two ("Candidatus Cyrtobacter" and "Candidatus Anadelfobacter") displayed an unusual pattern of competitive exclusion. These data represent the first insight into the prevalence and patterns of bacterial symbionts in natural populations of free-living protists.

Bacterial symbiosis in ciliates (Alveolata, Ciliophora): Roads traveled and those still to be taken.

The diversity of prokaryotic symbionts in Ciliophora and other protists is fascinatingly rich; they may even include some potentially pathogenic bacteria. In this review, we summarize currently available data on biodiversity and some morphological and biological peculiarities of prokaryotic symbionts mainly within the genera Paramecium and Euplotes. Another direction of ciliate symbiology, neglected for a long time and now re-discovered, is the study of epibionts of ciliates. This promises a variety of interesting outcomes. Last, but not least, we stress the new technologies, such as next generation sequencing and the use of genomics data, which all can clarify many new aspects of relevance. For this reason, a brief overview of achievements in genomic studies on ciliate's symbionts is provided. Summing up the results of numerous scientific contributions, we systematically update current knowledge and outline the prospects as to how symbiology of Ciliophora may develop in the near future.

Acrylate protects a marine bacterium from grazing by a ciliate predator.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

Characterization of a green Stentor with symbiotic algae growing in an extremely oligotrophic environment and storing large amounts of starch granules in its cytoplasm.

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1-3 µm refractive granules that turn brown by Lugol's staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.

Segnochrobactrum spirostomi gen. nov., sp. nov., isolated from the ciliate Spirostomum yagiui and description of a novel family, Segnochrobactraceae fam. nov. within the order Rhizobiales of the class Alphaproteobacteria.

A bacterial strain, designated Sp-1T, was isolated from the heterotrich ciliate Spirostomum yagiui collected from a reservoir located in Ulsan, Republic of Korea. Cells of Sp-1T were Gram stain-negative, rod-shaped, non-spore-forming, non-motile and contained poly-ß-hydroxybutyrate granules. Phylogenetic analyses based on 16S rRNA gene sequences indicated that Sp-1T constituted a distinct phylogenetic lineage within different families in the order Rhizobiales with a pairwise sequence similarity of 95 % to the species of the genus Ochrobactrum: Ochrobactrum anthropi ATCC 49188T and Ochrobactrum cytisi ESC1T (family Brucellaceae). The major cellular fatty acids were C19 : 0 cyclo ω8c (44.4 %) and C16 : 0 (32.1 %). The identified sole isoprenoid quinone was ubiquinone-10 (Q-10). The major polar lipids produced were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, two unidentified phospholipids and three unidentified lipids. The genome size was about 5.4 Mbp and the DNA G+C content was 68.2 mol%. Sp-1T exhibited the highest average nucleotide identity value of 76.6 % and in silico DNA-DNA hybridization value of 22.1 % with Pseudoxanthobacter soli DSM 19599T (family Xanthobacteraeae). This strain is distinguishable from closely related members of the order Rhizobiales by its differential phenotypic, chemotaxonomic, genomic and phylogenetic characteristics. On the basis of evidence from polyphasic taxonomic analysis, we concluded that Sp-1T represents a novel species in a novel genus within the order Rhizobiales, for which the name Segnochrobactrum spirostomi gen. nov., sp. nov. is proposed. The type strain is Sp-1T (=KCTC 62036T=JCM 32162T). We also describe a novel family, Segnochrobactraceae fam. nov., to encompass the proposed novel genus and species.

Tripartite Symbiosis of an Anaerobic Scuticociliate with Two Hydrogenosome-Associated Endosymbionts, a Holospora-Related Alphaproteobacterium and a Methanogenic Archaeon.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Thiotrophic bacterial symbiont induces polyphenism in giant ciliate host Zoothamnium niveum.

Evolutionary theory predicts potential shifts between cooperative and uncooperative behaviour under fluctuating environmental conditions. This leads to unstable benefits to the partners and restricts the evolution of dependence. High dependence is usually found in those hosts in which vertically transmitted symbionts provide nutrients reliably. Here we study host dependence in the marine, giant colonial ciliate Zoothamnium niveum and its vertically transmitted, nutritional, thiotrophic symbiont from an unstable environment of degrading wood. Previously, we have shown that sulphidic conditions lead to high host fitness and oxic conditions to low fitness, but the fate of the symbiont has not been studied. We combine several experimental approaches to provide evidence for a sulphide-tolerant host with striking polyphenism involving two discrete morphs, a symbiotic and an aposymbiotic one. The two differ significantly in colony growth form and fitness. This polyphenism is triggered by chemical conditions and elicited by the symbiont's presence on the dispersing swarmer. We provide evidence of a single aposymbiotic morph found in nature. We propose that despite a high fitness loss when aposymbiotic, the ciliate has retained a facultative life style and may use the option to live without its symbiont to overcome spatial and temporal shortage of sulphide in nature.

The core microbiome of sessile ciliate Stentor coeruleus is not shaped by the environment.

Microbiomes of multicellular organisms are one of the hottest topics in microbiology and physiology, while only few studies addressed bacterial communities associated with protists. Protists are widespread in all environments and can be colonized by plethora of different bacteria, including also human pathogens. The aim of this study was to characterize the prokaryotic community associated with the sessile ciliate Stentor coeruleus. 16S rRNA gene metabarcoding was performed on single cells of S. coeruleus and on their environment, water from the sewage stream. Our results showed that the prokaryotic community composition differed significantly between Stentor cells and their environment. The core microbiome common for all ciliate specimens analyzed could be defined, and it was composed mainly by representatives of bacterial genera which include also potential human pathogens and commensals, such as Neisseria, Streptococcus, Capnocytophaga, Porphyromonas. Numerous 16S rRNA gene contigs belonged to endosymbiont "Candidatus Megaira polyxenophila". Our data suggest that each ciliate cell can be considered as an ecological microniche harboring diverse prokaryotic organisms. Possible benefits for persistence and transmission in nature for bacteria associated with protists are discussed. Our results support the hypothesis that ciliates attract potentially pathogenic bacteria and play the role of natural reservoirs for them.

Sulfur-Oxidizing Symbionts without Canonical Genes for Autotrophic CO2 Fixation.

Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO2 by the symbionts drives these nutritional associations. In this study, we investigated "Candidatus Kentron," the clade of symbionts hosted by Kentrophoros, a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron bacteria lack the key canonical genes for any of the known pathways for autotrophic carbon fixation and have a carbon stable isotope fingerprint that is unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to gain energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. Here we show that Kentron bacteria are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or the reverse tricarboxylic acid cycle for autotrophy.IMPORTANCE Many animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO2, serving as primary producers for their hosts. Here we describe a clade of nonautotrophic sulfur-oxidizing symbionts, "Candidatus Kentron," associated with marine ciliates. They lack genes for known autotrophic pathways and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead, they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

Towards an ecological understanding of the killer trait - A reproducible protocol for testing its impact on freshwater ciliates.

Paramecium strains with the ability to kill other paramecia often harbour intracellular bacteria belonging to the genera Caedibacter or Caedimonas. Central structures of this killer trait are refractile bodies (R-bodies) produced by the endosymbionts. Once ingested by a sensitive Paramecium, R-bodies presumably act as delivery system for an unidentified toxin which causes the death of endosymbiont-free paramecia while those infected gain resistance from their symbionts. The killer trait is therefore considered as competitive advantage for the hosts of R-body producers. While its effectiveness against paramecia is well documented, the effects on other aquatic ciliates are much less studied. In order to address the broadness of the killer trait, a reproducible killer test assay considering the effects on predatory ciliates (Climacostomum virens and Dileptus jonesi) as well as potential bacterivorous Paramecium competitors (Dexiostoma campyla, Euplotes aediculatus, Euplotes woodruffi, and Spirostomum teres) as possibly susceptible species was established. All used organisms were molecularly characterized to increase traceability and reproducibility. The absence of any lethal effects in both predators and competitors after exposure to killer paramecia strongly suggests a narrow action range for the killer trait. Thus, R-body producing bacteria provide their host with a complex, costly strategy to outcompete symbiont-free congeners only.

Diversity and environmental distribution of the cosmopolitan endosymbiont "Candidatus Megaira".

Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont "Candidatus Megaira", which is phylogenetically closely related to the pathogen Rickettsia. "Candidatus Megaira" was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species "Candidatus Megaira polyxenophila", and the multidisciplinary description of a novel species, "Candidatus Megaira venefica", presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of "Candidatus Megaira", and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.

Genetic basis for the establishment of endosymbiosis in Paramecium.

The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria. The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria, and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

Detecting Associations Between Ciliated Protists and Prokaryotes with Culture-Independent Single-Cell Microbiomics: a Proof-of-Concept Study.

Symbioses between prokaryotes and microbial eukaryotes, particularly ciliated protists, have been studied for a long time. Nevertheless, researchers have focused only on a few host genera and species, mainly due to difficulties in cultivating the hosts, and usually have considered a single symbiont at a time. Here, we present a pilot study using a single-cell microbiomic approach to circumvent these issues. Unicellular ciliate isolation followed by simultaneous amplification of eukaryotic and prokaryotic markers was used. Our preliminary test gave reliable and satisfactory results both on samples collected from different habitats (marine and freshwater) and on ciliates belonging to different taxonomic groups. Results suggest that, as already assessed for many macro-organisms like plants and metazoans, ciliated protists harbor distinct microbiomes. The applied approach detected new potential symbionts as well as new hosts for previously described ones, with relatively low time and cost effort and without culturing. When further developed, single-cell microbiomics for ciliates could be applied to a large number of studies aiming to unravel the evolutionary and ecological meaning of these symbiotic systems.

Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution.

Evolution of the Arsenal of Legionella pneumophila Effectors To Modulate Protist Hosts.

Within the human host, Legionella pneumophila replicates within alveolar macrophages, leading to pneumonia. However, L. pneumophila is an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles of L. pneumophila within the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome of L. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors among Legionella species and >320 effector proteins in L. pneumophila into host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation of L. pneumophila to various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrict L. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure of L. pneumophila to overcome restriction of certain protist hosts. Here, we review the interaction of L. pneumophila with its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge of L. pneumophila-protist host interaction and L. pneumophila's evolution to become a human pathogen.

Genomes of two archaeal endosymbionts show convergent adaptations to an intracellular lifestyle.

Endosymbiosis is a widespread phenomenon in the microbial world and can be based on diverse interactions between endosymbiont and host cell. The vast majority of the known endosymbiotic interactions involve bacteria that have invaded eukaryotic host cells. However, methanogenic archaea have been found to thrive in anaerobic, hydrogenosome-containing protists and it was suggested that this symbiosis is based on the transfer of hydrogen. Here, we used culture-independent genomics approaches to sequence the genomes of two distantly related methanogenic endosymbionts that have been acquired in two independent events by closely related anaerobic ciliate hosts Nyctotherus ovalis and Metopus contortus, respectively. The sequences obtained were then validated as originating from the ciliate endosymbionts by in situ probing experiments. Comparative analyses of these genomes and their closest free-living counterparts reveal that the genomes of both endosymbionts are in an early stage of adaptation towards endosymbiosis as evidenced by the large number of genes undergoing pseudogenization. For instance, the observed loss of genes involved in amino acid biosynthesis in both endosymbiont genomes indicates that the endosymbionts rely on their hosts for obtaining several essential nutrients. Furthermore, the endosymbionts appear to have gained significant amounts of genes of potentially secreted proteins, providing targets for future studies aiming to elucidate possible mechanisms underpinning host-interactions. Altogether, our results provide the first genomic insights into prokaryotic endosymbioses from the archaeal domain of life.

Tropidoatractidae fam. nov., a Deep Branching Lineage of Metopida (Armophorea, Ciliophora) Found in Diverse Habitats and Possessing Prokaryotic Symbionts.

We report a discovery of a novel family of anaerobic ciliates, Tropidoatractidae fam. nov. Phylogenetic analyses based on the 18S rRNA gene show that the family Tropidoatractidae corresponds to the previously reported clade of environmental sequences closely related to the lineage consisting of orders Metopida and Clevelandellida. The family comprises two genera, Tropidoatractus and Palmarella, and five species, two of which are newly described herein. Tropidoatractidae are cosmopolitan Metopida with sparse somatic and oral ciliature, deep, cup-like buccal cavity, and hyaline cortex with interkinetal ridges. Moreover, all species occur in two morphotypes, slender and stout. They inhabit microoxic or anoxic freshwater, brackish, and marine sediments and possess anaerobic mitochondrion-related organelles and various prokaryotic symbionts. The discovery of Tropidoatractidae provides valuable information about the evolution of Armophorea and gives us insights to the diversity and ecological preferences of anaerobic ciliates in general.

The genome of an endosymbiotic methanogen is very similar to those of its free-living relatives.

The methanogenic endosymbionts of anaerobic protists represent the only known intracellular archaea, yet, almost nothing is known about genome structure and content in these lineages. Here, an almost complete genome of an intracellular Methanobacterium species was assembled from a metagenome derived from its host ciliate, a Heterometopus species. Phylogenomic analysis showed that the endosymbiont was closely related to free-living Methanobacterium isolates, and when compared with the genomes of free-living Methanobacterium, the endosymbiont did not show significant reduction in genome size or GC content. Additionally, the Methanobacterium endosymbiont genome shared the majority of its genes with its closest relative, though it did also contain unique genes possibly involved in interactions with the host via membrane-associated proteins, the removal of toxic by-products from host metabolism and the production of small signalling molecules. Though anaerobic ciliates have been shown to transmit their endosymbionts to daughter cells during division, the results presented here could suggest that the endosymbiotic Methanobacterium did not experience significant genetic isolation or drift and/or that this lineage was only recently acquired. Altogether, comparative genomic analysis identified genes potentially involved in the establishment and maintenance of the symbiosis, as well provided insight into the genomic consequences for an intracellular archaeum.

Symbiont replacement between bacteria of different classes reveals additional layers of complexity in the evolution of symbiosis in the ciliate Euplotes.

Symbiosis is a diverse and complex phenomenon requiring diverse model systems. The obligate relationship between a monophyletic group of Euplotes species ("clade B") and the betaproteobacteria Polynucleobacter and "Candidatus Protistobacter" is among the best-studied in ciliates, and provides a framework to investigate symbiont replacements. Several other Euplotes-bacteria relationships exist but are less understood, such as the co-dependent symbiosis between Euplotes magnicirratus (which belongs to "clade A") and the alphaproteobacterium "Candidatus Devosia euplotis". Here we describe a new Devosia inhabiting the cytoplasm of a strain of Euplotes harpa, a clade B species that usually depends on Polynucleobacter for survival. The novel bacterial species, "Candidatus Devosia symbiotica", is closely related to the symbiont of E. magnicirratus, casting a different light on the history of bacteria colonizing ciliates of this genus. The two Devosia species may have become symbionts independently or as the result of a symbiont exchange between hosts, in either case replacing a previous essential bacterium in E. harpa. Alternatively, both may be remnants of an ancient symbiotic relationship between Euplotes and Devosia, in which case Polynucleobacter and "Ca. Protistobacter" are recent invaders. Either way, symbiont replacement between bacteria belonging to different classes must be evoked to explain this fascinating system.