'Candidatus Gromoviella agglomerans', a novel intracellular Holosporaceae parasite of the ciliate Paramecium showing marked genome reduction.

Holosporales are an alphaproteobacterial lineage encompassing bacteria obligatorily associated with multiple diverse eukaryotes. For most representatives, little is known on the interactions with their hosts. In this study, we characterized a novel Holosporales symbiont of the ciliate Paramecium polycaryum. This bacterium inhabits the host cytoplasm, frequently forming quite large aggregates. Possibly due to such aggregates, host cells sometimes displayed lethal division defects. The symbiont was also able to experimentally stably infect another Paramecium polycaryum strain. The bacterium is phylogenetically related with symbionts of other ciliates and diplonemids, forming a putatively fast-evolving clade within the family Holosporaceae. Similarly to many close relatives, it presents a very small genome (<600 kbp), and, accordingly, a limited predicted metabolism, implying a heavy dependence on Paramecium, thanks also to some specialized membrane transporters. Characterized features, including the presence of specific secretion systems, are overall suggestive of a mild parasitic effect on the host. From an evolutionary perspective, a potential ancestral trend towards pronounced genome reduction and possibly linked to parasitism could be inferred, at least among fast-evolving Holosporaceae, with some lineage-specific traits. Interestingly, similar convergent features could be observed in other host-associated lineages, in particular Rickettsiales among Alphaproteobacteria.

Clarification of the Taxonomic Position of Paramecium caudatum Micronucleus Symbionts.

Bacteria of genus Holospora (order Holosporales, class Alphaproteobacteria) are obligate intranuclear symbionts of ciliates Paramecium spp. with strict host species and nuclear (macronucleus or micronucleus) specificity. However, three species under study Holospora undulata, Holospora elegans and 'Holospora recta' occupy the same ecological niche-micronucleus of Paramecium caudatum and demonstrate some differences in morphology of infectious form. The genetic diversity of holosporas by rrs and rpoB sequence analysis was determined. Phylogenetic and phylogenomic analysis of Holospora spp., as well as some phenotypic features indicate that there is no distinctive difference supporting studied micronuclear endosymbionts as distinct species. Therefore, Holospora elegans and 'Holospora recta' should be considered subspecies of Holospora undulata (ex Haffkine 1890) Gromov and Ossipov 1981, which was described first. Thus, we confirmed the evolutionary aspects of the development of symbiotic relationships: holosporas have a strict specificity to the host species and the type of nucleus.

Epidemiology of Nucleus-Dwelling Holospora: Infection, Transmission, Adaptation, and Interaction with Paramecium.

The chapter describes the exceptional symbiotic associations formed between the ciliate Paramecium and Holospora, highly infectious bacteria residing in the host nuclei. Holospora and Holospora-like bacteria (Alphaproteobacteria) are characterized by their ability for vertical and horizontal transmission in host populations, a complex biphasic life cycle, and pronounced preference for host species and colonized cell compartment. These bacteria are obligate intracellular parasites; thus, their metabolic repertoire is dramatically reduced. Nevertheless, they perform complex interactions with the host ciliate. We review ongoing efforts to unravel the molecular adaptations of these bacteria to their unusual lifestyle and the host's employment in the symbiosis. Furthermore, we summarize current knowledge on the genetic and genomic background of Paramecium-Holospora symbiosis and provide insights into the ecological and evolutionary consequences of this interaction. The diversity and occurrence of symbioses between ciliates and Holospora-like bacteria in nature is discussed in connection with transmission modes of symbionts, host specificity and compatibility of the partners. We aim to summarize 50 years of research devoted to these symbiotic systems and conclude trying to predict some perspectives for further studies.

Highly Reduced Genomes of Protist Endosymbionts Show Evolutionary Convergence.

Genome evolution in bacterial endosymbionts is notoriously extreme: the combined effects of strong genetic drift and unique selective pressures result in highly reduced genomes with distinctive adaptations to hosts [1-4]. These processes are mostly known from animal endosymbionts, where nutritional endosymbioses represent the best-studied systems. However, eukaryotic microbes, or protists, also harbor diverse bacterial endosymbionts, but their genome reduction and functional relationships with their hosts are largely unexplored [5-7]. We sequenced the genomes of four bacterial endosymbionts from three species of diplonemids, poorly studied but abundant and diverse heterotrophic protists [8-12]. The endosymbionts come from two bacterial families, Rickettsiaceae and Holosporaceae, that have invaded two families of diplonemids, and their genomes have converged on an extremely small size (605-632 kilobase pairs [kbp]), similar gene content (e.g., metabolite transporters and secretion systems), and reduced metabolic potential (e.g., loss of energy metabolism). These characteristics are generally found in both families, but the diplonemid endosymbionts have evolved greater extremes in parallel. They possess modified type VI secretion systems that could function in manipulating host metabolism or other intracellular interactions. Finally, modified cellular machinery like the ATP synthase without oxidative phosphorylation, and the reduced flagellar apparatus present in some diplonemid endosymbionts and nutritional animal endosymbionts, indicates that intracellular mechanisms have converged in bacterial endosymbionts with various functions and from different eukaryotic hosts across the tree of life.

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.

"Candidatus Hafkinia simulans" gen. nov., sp. nov., a Novel Holospora-Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host.

We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, "Candidatus Hafkinia simulans", was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra's phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp.

The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum, and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.

Complex life cycle, broad host range and adaptation strategy of the intranuclear Paramecium symbiont Preeria caryophila comb. nov.

Holospora and related bacteria are a group of obligate Paramecium symbionts. Characteristic features are their infectivity, the presence of two distinct morphotypes, and usually a strict specialization for a single Paramecium species as host and for a nuclear compartment (either somatic or generative nucleus) for reproduction. Holospora caryophila steps out of line, naturally occurring in Paramecium biaurelia and Paramecium caudatum. This study addresses the phylogenetic relationship among H. caryophila and other Holospora species based on 16S rRNA gene sequence comparison analyzing the type strain and seven new macronuclear symbionts. Key aspects of Holospora physiology such as infectivity, symbiosis establishment and host range were determined by comprehensive infection assays. Detailed morphological investigations and sequence-based phylogeny confirmed a high similarity between the type strain of H. caryophila and the novel strains. Surprisingly, they are only distantly related to other Holospora species suggesting that they belong to a new genus within the family Holosporaceae, here described as Preeria caryophila comb. nov. Adding to this phylogenetic distance, we also observed a much broader host range, comprising at least eleven Paramecium species. As these potential host species exhibit substantial differences in frequency of sexual processes, P. caryophila demonstrates which adaptations are crucial for macronuclear symbionts facing regular destruction of their habitat.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium "Candidatus Holospora parva".

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.

Draft genome sequences of three Holospora species (Holospora obtusa, Holospora undulata, and Holospora elegans), endonuclear symbiotic bacteria of the ciliate Paramecium caudatum.

We present draft genome sequences of three Holospora species, hosted by the ciliate Paramecium caudatum; that is, the macronucleus-specific H. obtusa and the micronucleus-specific H. undulata and H. elegans. We investigate functions of orthologous core genes conserved across the three Holospora species, which may be essential for the infection and survival in the host nucleus.

Phylogenetic placement of two previously described intranuclear bacteria from the ciliate Paramecium bursaria (Protozoa, Ciliophora): 'Holospora acuminata' and 'Holospora curviuscula'.

'Holospora acuminata' infects micronuclei of Paramecium bursaria (Protozoa, Ciliophora), whereas 'Holospora curviuscula' infects the macronucleus in other clones of the same host species. Because these micro-organisms have not been cultivated, their description has been based only on some morphological properties and host and nuclear specificities. One16S rRNA gene sequence of 'H. curviuscula' is present in databases. The systematic position of the representative strain of 'H. curviuscula', strain MC-3, was determined in this study. Moreover, for the first time, two strains of 'H. acuminata', KBN10-1 and AC61-10, were investigated. Phylogenetic analysis indicated that all three strains belonged to the genus Holospora, family Holosporaceae, order Rickettsiales within the Alphaproteobacteria.

Revised systematics of Holospora-like bacteria and characterization of "Candidatus Gortzia infectiva", a novel macronuclear symbiont of Paramecium jenningsi.

The genus Holospora (Rickettsiales) includes highly infectious nuclear symbionts of the ciliate Paramecium with unique morphology and life cycle. To date, nine species have been described, but a molecular characterization is lacking for most of them. In this study, we have characterized a novel Holospora-like bacterium (HLB) living in the macronuclei of a Paramecium jenningsi population. This bacterium was morphologically and ultrastructurally investigated in detail, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and developed a specific probe for fluorescence in situ hybridization experiments. A new taxon, "Candidatus Gortzia infectiva", was established for this HLB according to its unique characteristics and the relatively low DNA sequence similarities shared with other bacteria. The phylogeny of the order Rickettsiales based on 16S rRNA gene sequences has been inferred, adding to the available data the sequence of the novel bacterium and those of two Holospora species (Holospora obtusa and Holospora undulata) characterized for the purpose. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and showed a possible pattern of evolution for some of their features. We suggested to classify inside the family Holosporaceae only HLBs, excluding other more distantly related and phenotypically different Paramecium endosymbionts.

"Candidatus Paraholospora nucleivisitans", an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host.

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological-botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.

Micronucleus-specific bacterium Holospora elegans irreversibly enhances stress gene expression of the host Paramecium caudatum.

The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear-specific symbiont Holospora obtusa survived better than symbiont-free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 degrees C, a usual growth temperature. We report herein that paramecia bearing the micronuclear-specific symbiont Holospora elegans also acquire the heat-shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock-resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 degrees C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat-shock resistance.

Direct and correlated responses to selection in a host-parasite system: testing for the emergence of genotype specificity.

Genotype x environment interactions can facilitate coexistence of locally adapted specialists. Interactions evolve if adaptation to one environment trades off with performance in others. We investigated whether evolution on one host genotype traded off with performance on others in long-term experimental populations of different genotypes of the protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. A total of nine parasite selection lines evolving on three host genotypes and the ancestral parasite were tested in a cross-infection experiment. We found that evolved parasites produced more infections than did the ancestral parasites, both on host genotypes they had evolved on (positive direct response to selection) and on genotypes they had not evolved on (positive correlated response to selection). On two host genotypes, a negative relationship between direct and correlated responses indicated pleiotropic costs of adaptation. On the third, a positive relationship suggested cost-free adaptation. Nonetheless, on all three hosts, resident parasites tended to be superior to the average nonresident parasite. Thus genotype specificity (i.e., patterns of local adaptation) may evolve without costs of adaptation, as long as direct responses to selection exceed correlated responses.

Experimental evolution of resistance in Paramecium caudatum against the bacterial parasite Holospora undulata.

Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by frequency-dependent selection. This requires that different parasite genotypes perform differently on different host genotypes. Such genotype-by-genotype interactions arise if adaptation to one host (or parasite) genotype reduces performance on others. These direct costs of adaptation can maintain genetic polymorphism and generate geographic patterns of local host or parasite adaptation. Fixation of all-resistant (or all-infective) genotypes is further prevented if adaptation trades off with other host (or parasite) life-history traits. For the host, such indirect costs of resistance refer to reduced fitness of resistant genotypes in the absence of parasites. We studied (co)evolution in experimental microcosms of several clones of the freshwater protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. After two and a half years of culture, inoculation of evolved and naive (never exposed to the parasite) hosts with evolved and founder parasites revealed an increase in host resistance, but not in parasite infectivity. A cross-infection experiment showed significant host clone-by-parasite isolate interactions, and evolved hosts tended to be more resistant to their own (local) parasites than to parasites from other hosts. Compared to naive clones, evolved host clones had lower division rates in the absence of the parasite. Thus, our study indicates de novo evolution of host resistance, associated with both direct and indirect costs. This illustrates how interactions with parasites can lead to the genetic divergence of initially identical populations.