Comparative genomic analysis of symbiotic and free-living Fluviibacter phosphoraccumulans strains provides insights into the evolutionary origins of obligate Euplotes-bacterial endosymbioses.

Endosymbiosis is a widespread and important phenomenon requiring diverse model systems. Ciliates are a widespread group of protists that often form symbioses with diverse microorganisms. Endosymbioses between the ciliate Euplotes and heritable bacterial symbionts are common in nature, and four essential symbionts were described: Polynucleobacter necessarius, "Candidatus Protistobacter heckmanni," "Ca. Devosia symbiotica," and "Ca. Devosia euplotis." Among them, only the genus Polynucleobacter comprises very close free-living and symbiotic representatives, which makes it an excellent model for investigating symbiont replacements and recent symbioses. In this article, we characterized a novel endosymbiont inhabiting the cytoplasm of Euplotes octocarinatus and found that it is a close relative of the free-living bacterium Fluviibacter phosphoraccumulans (Betaproteobacteria and Rhodocyclales). We present the complete genome sequence and annotation of the symbiotic Fluviibacter. Comparative analyses indicate that the genome of symbiotic Fluviibacter is small in size and rich in pseudogenes when compared with free-living strains, which seems to fit the prediction for recently established endosymbionts undergoing genome erosion. Further comparative analysis revealed reduced metabolic capacities in symbiotic Fluviibacter, which implies that the symbiont relies on the host Euplotes for carbon sources, organic nitrogen and sulfur, and some cofactors. We also estimated substitution rates between symbiotic and free-living Fluviibacter pairs for 233 genes; the results showed that symbiotic Fluviibacter displays higher dN/dS mean value than free-living relatives, which suggested that genetic drift is the main driving force behind molecular evolution in endosymbionts. IMPORTANCE: In the long history of symbiosis research, most studies focused mainly on organelles or bacteria within multicellular hosts. The single-celled protists receive little attention despite harboring an immense diversity of symbiotic associations with bacteria and archaea. One subgroup of the ciliate Euplotes species is strictly dependent on essential symbionts for survival and has emerged as a valuable model for understanding symbiont replacements and recent symbioses. However, almost all of our knowledge about the evolution and functions of Euplotes symbioses comes from the Euplotes-Polynucleobacter system. In this article, we report a novel essential symbiont, which also has very close free-living relatives. Genome analysis indicated that it is a recently established endosymbiont undergoing genome erosion and relies on the Euplotes host for many essential molecules. Our results provide support for the notion that essential symbionts of the ciliate Euplotes evolve from free-living progenitors in the natural water environment.

Bipolar Biogeographical Distribution of Parafrancisella Bacteria Carried by the Ciliate Euplotes.

Parafrancisella adeliensis, a Francisella-like endosymbiont, was found to reside in the cytoplasm of an Antarctic strain of the bipolar ciliate species, Euplotes petzi. To inquire whether Euplotes cells collected from distant Arctic and peri-Antarctic sites host Parafrancisella bacteria, wild-type strains of the congeneric bipolar species, E. nobilii, were screened for Parafrancisella by in situ hybridization and 16S gene amplification and sequencing. Results indicate that all Euplotes strains analyzed contained endosymbiotic bacteria with 16S nucleotide sequences closely similar to the P. adeliensis 16S gene sequence. This finding suggests that Parafrancisella/Euplotes associations are not endemic to Antarctica, but are common in both the Antarctic and Arctic regions.

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.

All essential endosymbionts of the ciliate Euplotes are cyclically replaced.

Symbiotic systems vary in the degree to which the partners are bound to each other1. At one extreme, there are intracellular endosymbionts in mutually obligate relationships with their host, often interpreted as mutualistic. The symbiosis between the betaproteobacterium Polynucleobacter and the ciliate Euplotes (clade B) challenges this view2: although freshwater Euplotes species long ago became dependent on endosymbionts, the many extant Polynucleobacter lineages they harbour arose recently and in parallel from different free-living ancestors2. The host requires the endosymbionts for reproduction and survival3, but each newly established symbiont is ultimately driven to extinction in a cycle of establishment, degeneration, and replacement. Similar replacement events have been observed in sap-feeding insects4-6, a model for bacteria-eukaryote symbioses7, but usually only affect a small subset of the host populations. Most insects retain an ancient coevolving symbiont, suggesting that long-term mutualism and permanent integration remain the rule and symbiont turnovers are mere evolutionary side-stories. Here we show that this is not the case for Euplotes. We examined all known essential Euplotes symbionts and found that none are ancient or coevolving; rather, all are recently established and continuously replaced over relatively short evolutionary time spans, making the symbiosis ancient for the host but not for any bacterial lineage.

Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Morphology, ultrastructure, genomics, and phylogeny of Euplotes vanleeuwenhoeki sp. nov. and its ultra-reduced endosymbiont "Candidatus Pinguicoccus supinus" sp. nov.

Taxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as "holobionts". We illustrate this approach through the description of the ciliate Euplotes vanleeuwenhoeki sp. nov. and its bacterial endosymbiont "Candidatus Pinguicoccus supinus" gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.

Synthesis of Bioactive Silver Nanoparticles by a Pseudomonas Strain Associated with the Antarctic Psychrophilic Protozoon Euplotes focardii.

The synthesis of silver nanoparticles (AgNPs) by microorganisms recently gained a greater interest due to its potential to produce them in various sizes and morphologies. In this study, for AgNP biosynthesis, we used a new Pseudomonas strain isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii. After incubation of Pseudomonas cultures with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. Scanning electron (SEM) and transmission electron microscopy (TEM) revealed spherical polydispersed AgNPs in the size range of 20-70 nm. The average size was approximately 50 nm. Energy dispersive X-ray spectroscopy (EDS) showed the presence of a high intensity absorption peak at 3 keV, a distinctive property of nanocrystalline silver products. Fourier transform infrared (FTIR) spectroscopy found the presence of a high amount of AgNP-stabilizing proteins and other secondary metabolites. X-ray diffraction (XRD) revealed a face-centred cubic (fcc) diffraction spectrum with a crystalline nature. A comparative study between the chemically synthesized and Pseudomonas AgNPs revealed a higher antibacterial activity of the latter against common nosocomial pathogen microorganisms, including Escherichia coli, Staphylococcus aureus and Candida albicans. This study reports an efficient, rapid synthesis of stable AgNPs by a new Pseudomonas strain with high antimicrobial activity.

Symbionts of the ciliate Euplotes: diversity, patterns and potential as models for bacteria-eukaryote endosymbioses.

Endosymbioses between bacteria and eukaryotes are enormously important in ecology and evolution, and as such are intensely studied. Despite this, the range of investigated hosts is narrow in the context of the whole eukaryotic tree of life: most of the information pertains to animal hosts, while most of the diversity is found in unicellular protists. A prominent case study is the ciliate Euplotes, which has repeatedly taken up the bacterium Polynucleobacter from the environment, triggering its transformation into obligate endosymbiont. This multiple origin makes the relationship an excellent model to understand recent symbioses, but Euplotes may host bacteria other than Polynucleobacter, and a more detailed knowledge of these additional interactions is needed in order to correctly interpret the system. Here, we present the first systematic survey of Euplotes endosymbionts, adopting a classical as well as a metagenomic approach, and review the state of knowledge. The emerging picture is indeed quite complex, with some Euplotes harbouring rich, stable prokaryotic communities not unlike those of multicellular animals. We provide insights into the distribution, evolution and diversity of these symbionts (including the establishment of six novel bacterial taxa), and outline differences and similarities with the most well-understood group of eukaryotic hosts: insects.

A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.

Parallel genome reduction in symbionts descended from closely related free-living bacteria.

Endosymbiosis plays an important role in ecology and evolution, but fundamental aspects of the origin of intracellular symbionts remain unclear. The extreme age of many symbiotic relationships, lack of data on free-living ancestors and uniqueness of each event hinder investigations. Here, we describe multiple strains of the bacterium Polynucleobacter that evolved independently and under similar conditions from closely related, free-living ancestors to become obligate endosymbionts of closely related ciliate hosts. As these genomes reduced in parallel from similar starting states, they provide unique glimpses into the mechanisms underlying genome reduction in symbionts. We found that gene loss is contingently lineage-specific, with no evidence for ordered streamlining. However, some genes in otherwise disrupted pathways are retained, possibly reflecting cryptic genetic network complexity. We also measured substitution rates between many endosymbiotic and free-living pairs for hundreds of genes, which showed that genetic drift, and not mutation pressure, is the main non-selective factor driving molecular evolution in endosymbionts.

High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora, Euplotia).

The Betaproteobacteria-Euplotes association is an obligatory symbiotic system involving a monophyletic group of ciliate species and two betaproteobacteria species which can be alternatively present. Recent data showed that this relationship has been established more than once and that several symbiont-substitution events took place, revealing a complex and intriguing evolutionary path. Due to the different evolutionary pathways followed by the different symbionts, each bacterial strain could have differentially evolved and/or lost functional traits. Therefore, we performed re-infection experiments, both by phagocytosis and by microinjection, to test the possible functional role of the different bacteria towards the ciliates. Our results confirm that the growth capacity of the host is indissolubly linked to the presence of its original symbionts. Results of the attempts of re-infection by phagocytosis showed that none of the bacteria is able to successfully colonize the host cytoplasm in this way, even if regularly ingested. Re-infection by microinjection succeed only in one case. Such results point to a high degree of specificity in the interactions between bacteria and Euplotes even after the invasion step. Due to a co-evolutive pathway of reciprocal adaptation, different degree of re-colonization ability could have been conserved by the different species and strains of the symbionts.

A House for Two--Double Bacterial Infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) Sampled in Southeastern Brazil.

Several ciliated protists form symbiotic associations with a diversity of microorganisms, leading to drastic impact on their ecology and evolution. In this work, two Euplotes spp. sampled in Rio de Janeiro, Brazil, were identified based on morphological and molecular features as Euplotes woodruffi strain Sq1 and E. encysticus strain Sq2 and investigated for the presence of endosymbionts. While E. woodruffi Sq1 stably hosts two bacterial populations, namely Polynucleobacter necessarius (Betaproteobacteria) and a new member of the family "Candidatus Midichloriaceae" (Alphaproteobacteria, Rickettsiales), here described as "Candidatus Bandiella woodruffii," branching with a broad host range bacterial group found in association with cnidarians, sponges, euglenoids, and some arthropods; in E. encysticus Sq2 no symbiotic bacterium could be detected. The dispersion ability of this novel bacterium was tested by co-incubating E. woodruffi Sq1 with three different ciliate species. Among the tested strains "Ca. B. woodruffii" could only be detected in association with E. encysticus Sq2 with a prevalence of 20 % after 1 week and 40 % after 2 weeks, maintaining this level for up to 6 months. Nevertheless, this apparent in vitro association was abolished when E. woodruffi Sq1 donor was removed from the microcosm, suggesting that this bacterium has the capacity for at least a short-term survival outside its natural host and the aptitude to ephemerally interact with other organisms. Together, these findings strongly suggest the need for more detailed investigations to evaluate the host range for "Ca. B. woodruffii" and any possible pathogenic effect of this bacterium on other organisms including humans.

Evaluation of the sensitivity to zinc of ciliates Euplotes vannus and Euplotes crassus and their naturally associated bacteria isolated from a polluted tropical bay.

The aim of this study was to evaluate the Zn sensitivity of Euplotes vannus, Euplotes crassus, and their naturally associated bacteria sampled from sediments in the northwest and east regions of Guanabara Bay. The unexposed ciliates and bacteria did not appear to be negatively affected by 96 h of assay. In the control group, E. vannus exhibited an increase in the biomass content from 2.3 × 10(2) to 2.3 × 10(3) µg C cm(-3) between 0 and 96 h, and E. crassus increased up to 7.07 × 10(2) µg C cm(-3) at 48 h. The maximum biomass was pointed by E. crassus (1.33 × 10(3) µg C cm(-3)) in the presence of 0.005 mg Zn L(-1) and E. vannus was naturally associated bacteria (2.40 × 10(-1) µg C cm(-3)) in the presence of 1.0 mg Zn L(-1) (96 h). The growth of E. vannus from the northwest region showed concentration-dependent manners, and it is more sensitive to zinc than E. crassus from the southeast. Naturally associated bacteria showed better adaptation to increasing concentrations of Zn, and the Dunnett test showed that previous environmental selection is important. These results show that new bioremediation tools are necessary.

Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria.

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

"Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii", two novel ciliate endosymbionts belonging to the "Midichloria clade".

The "Midichloria clade" is a recently discovered but well-established evolutionary lineage clustering inside the order Rickettsiales (Alphaproteobacteria). Not much is known about the biology of these organisms. The best characterized ones are endocellular symbionts of very different eukaryotic hosts, ranging from arthropods to protists. "Candidatus Midichloria mitochondrii", the most studied organism of the group, is an interesting object of study because of its unique capability to infect metazoans' mitochondria and the presence of flagellar genes in its genome. With this work, we aim at increasing the knowledge on the biodiversity and phylogeny of the "Midichloria group". We characterized according to the "full cycle rRNA approach" two novel endosymbionts of ciliated protozoa, i.e. Paramecium nephridiatum and Euplotes aediculatus. According to the nomenclatural rules for uncultivated prokaryotes, we established the novel taxa "Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii" for the two bacterial symbionts. Our phylogenetic analysis based on 16S rRNA gene sequences confirms that the evolutionary histories of "Midichloria clade" representatives and of their hosts are very different. This suggests that the symbiotic processes arose many times independently, perhaps through ways of transmission still not described in Rickettsiales.

Characterization of "Candidatus Nebulobacter yamunensis" from the cytoplasm of Euplotes aediculatus (Ciliophora, Spirotrichea) and emended description of the family Francisellaceae.

Our knowledge of ciliate endosymbionts occurrence and diversity greatly expanded in the last decades, due to the development of characterization methods for uncultivable bacteria. Symbionts related to human pathogens such as rickettsiae and francisellae have been detected inside the cytoplasm of different ciliate species. In the present work, we have characterized a novel Francisella-related bacterium inside the rich prokaryotic community harbored by a population of Euplotes aediculatus (Ciliophora, Spirotrichea). Following the "Full-Cycle rRNA Approach" we obtained the almost full-length 16S rRNA gene sequence of this bacterium, and developed probes for diagnostic fluorescence in situ hybridizations. Attempts to culture the endosymbiont outside of its host failed. We classified this novel organism in a new taxon for which we propose the name "Candidatus Nebulobacter yamunensis". In order to investigate its evolutionary relationships, we have also performed phylogenetic analyses on the class Gammaproteobacteria and the order Thiotrichales, which include the monogeneric family Francisellaceae. We found highly supported evidences for the establishment of a new monophyletic taxon including Francisella species, other organisms currently incertae sedis, and "Candidatus Nebulobacter yamunensis". These organisms form a clade sharing a signature sequence not present in other Thiotrichales bacteria. Moreover, most of them have developed an intracellular life cycle inside eukaryotic organisms. We emended the original description of family Francisellaceae in order to encompass all members of the described clade.

Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association.

The Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of ciliate species belonging to the genus Euplotes and bacteria of the species Polynucleobacter necessarius (Betaproteobacteria). Both organisms are unable to survive independently. Several studies revealed the existence of free-living populations of Polynucleobacter bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Hence, following the most parsimonious explanation on the origin of the association, this symbiosis should represent a synapomorphic character for the hosts' clade. Nevertheless, phylogenetic analyses performed on an increased number of strains here presented suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in three cases, we observed different bacteria as obligate symbionts. These symbionts are the first characterized representatives of a phylogenetic lineage branching in a basal position with respect to the genus Polynucleobacter. The hypothesis that the original obligate symbionts belonged to this newly discovered clade and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria recruited from the environment is proposed and discussed. The evolutionary path of this association seems anyway to have been more complex than so far supposed.

Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi.

Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as "Candidatus F. noatunensis subsp. endociliophora" subsp. nov.

"Candidatus anadelfobacter veles" and "Candidatus cyrtobacter comes," two new rickettsiales species hosted by the protist ciliate Euplotes harpa (Ciliophora, Spirotrichea).

The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, "Candidatus Midichloria mitochondrii," as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the "Candidatus Midichloria" clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the "Candidatus Midichloria" clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the "Candidatus Midichloria" clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the "Candidatus Midichloria" clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, "Candidatus Anadelfobacter veles" and "Candidatus Cyrtobacter comes."

Emended description of the genus Polynucleobacter and the species Polynucleobacter necessarius and proposal of two subspecies, P. necessarius subsp. necessarius subsp. nov. and P. necessarius subsp. asymbioticus subsp. nov.

Heckmann and Schmidt described the genus Polynucleobacter for bacterial endosymbionts of freshwater ciliates affiliated with the genus Euplotes, and the species Polynucleobacter necessarius for obligate endosymbionts living in the cytoplasm of Euplotes aediculatus. Pure cultures of the type strain could not be established due to the obligate nature of the symbiotic relationship between the endosymbionts and their hosts. Therefore, Polynucleobacter necessarius is one of a few bacterial species with validly published names that lack a deposited pure culture. Meanwhile, it was demonstrated that the endosymbionts used for the description of the type of Polynucleobacter necessarius are closely related to obligately free-living strains. Similarity values of the 16S rRNA gene sequences obtained from the endosymbionts in the ciliate culture and free-living isolates in the range 99.1-99.4% indicate that these organisms belong to the same species. Here, we have emended the description of Polynucleobacter necessarius by characterization of free-living strains maintained as pure cultures. The species Polynucleobacter necessarius was characterized as having low G+C contents of the DNA (44-46 mol%), small genome sizes (1.5-2.5 Mbp) and a lack of motility. Because of distinct differences in lifestyle and the genome size of Polynucleobacter necessarius strains, we propose that two novel subspecies should be established, Polynucleobacter necessarius subsp. necessarius subsp. nov. [with a type, which is a description based on endosymbionts in the culture 'stock 15' of the ciliate E. aediculatus (ATCC 30859)] and Polynucleobacter necessarius subsp. asymbioticus subsp. nov. [with the type strain QLW-P1DMWA-1T (=DSM 18221T=CIP 109841T)], for the obligate endosymbionts of E. aediculatus and Euplotes harpa and obligately free-living strains, respectively.