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Verrucomicrobiales are a newly proposed, deep branched division of bacteria from which only a handful of cultured isolates are available. Culture-independent analysis of environmental samples shows that members of Verrucomicrobiales are widespread and can be divided into several subdivisions. Symbiotic species of Euplotidium (Ciliophora) and Nematodes, epixenosomes and Xiphinemobacter spp. respectively, are according to the 16S sequences also members of this group. Up to now, only minimal sequence data mainly focused on 16S rRNA gene sequences are available for this group of organisms. The phylogenetic relatives of Verrucomicrobiales are still unclear with Chlamydiales and Planctomycetes being the most probable candidates. The present study describes preliminary results on phylogenetic relationships of Verrucomicrobiales by using protein-coding genes like Hsp70 and RpoC/B. The species which are used for this study are Prosthecobacter vanneervenii, P. debontii, Opitutus sp.VeGlc2, O. terrae and epixenosomes. The genes were amplified with designed consensus-degenerated primers and results are available on the Hsp70. The sequences show several differences within the group. Verrucomicrobium and Prosthecobacter sequences cluster together, but in P. debontii, two sequences were found, and the second one appears quite divergent. Also the two Opitutus genes are quite atypical and cluster together in a deep position. Preliminary attempt was performed on Euplotidium-epixenosome complex. However, amplified and cloned sequences revealed to be closely related to ciliate sequences. Further attempt will be performed applying microtiter-plate-subtractive-hybridization to characterize epixenosome genes.

Tubulins are typical eucaryotic genes, which have recently been found in the free-living bacteria Prosthecobacter (Verrucomicrobiales). Nevertheless, microtubule-like structures were never observed in Prostecobacter, whereas they were reported in epixenosomes which are symbiotic Verrucomicrobiales of the ciliate Euplotidium. In the present work, an extensive search for tubulin like genes was performed with several combinations of PCR-primers, designed according to different criteria. Although some combinations of these primers are able to amplify simultaneously eukaryotic α- and β-tubulin as well as Prosthecobacter tubulin genes, no bacterial-like tubulin genes were identified in other Verrucomicrobiales species. Because of host derived contaminations, even a subtractive PCR approach, using microtiter-plate-subtractive-hybridization, was used with the epixenosomes. The characterization of the genomic environment of the Prosthecobacter tubulin genes was performed by gene walking. The sequences obtained from P. debontii revealed a so-far undiscovered gene duplication. The bacterial tubulin operon looks conserved among Prosthecobacter species and includes an α-tubulin, a β-tubulin and a kinesin like protein. Upstream of two operons a repetetive sequence could be found which could be a hint for horizontal gene transfer. The analysis of the genomic environments of the tubulin genes provided open reading frames, which have homology to Verrucomicrobium spinosum genes (ongoing genome project data), whereas up to now the blast search did not reveal tubulin-like genes in this organism. Present results suggest, that the Prosthecobacter tubulin genes are possibly of recent origin, maybe acquired by horizontal gene transfer, or lost by other so far studied Verrucomicrobiales. The origin and nature of microtubular genetic determinants from epixenosomes remains open.

Four different associations between ciliates of the family Euplotidae and bacteria have been studied. The ectosymbiotic relationship between ciliates of the genus Euplotidium and epixenosomes, (bacteria related to Verrucomicrobia), offers cues for ecological and evolutionary studies. Epixenosomes, although not vital for their host, confer on them a selective advantage, i.e. defense against predation. Epixenosomes have a compartmentalized cell and possess microtubule-like structures. Interestingly, the presence of two tubulin-like genes has been reported in members of the free-living verrucomicrobial genus Prosthecobacter. We obtained evidence that well-known Polynucleobacter bacteria, upon which a group of fresh-water Euplotes species depends, interfere with the glycogen metabolism of their hosts. Euplotes magicirratus is no longer able to digest food-organisms and is destined to die, once deprived of its specific prokaryotic endosymbiont, likely a new species of the alpha-proteobacterial genus Devosia. A further intriguing finding is the regular presence of rickettsiae in the cytoplasm of a marine Diophrys species. This is the first report of these bacteria in protozoa. This short list suggests that specialized mutualism is more widespread in the phylum than so far supposed.

Epixenosomes live on the dorsal surface of their ciliate host,Euplotidium itoi. They lack a nuclear envelope and divide like prokaryotes. On the other hand they have a morphological and functional cell compartmentalization and possess tubules that are sensible to tubulin inhibitors and positively react with different antitubulin antibodies. In the present paper, as a first step to investigate their real nature, the in situ hybridization technique was applied at the ultrastructural level. Different prokaryotic and eukaryotic probes suitable for detecting rRNA genes were used. An additional test was performed with the gene encoding for β tubulin in the ciliateEuplotes crassus. Positive results, evidenced by a precise localization of gold particles, were obtained with all the eukaryotic probes used. These probes were obtained from organisms belonging to three different kingdoms (Protista, Animalia, Plantae). On the contrary, no hybridization was obtained with prokaryotic probes, not even when the probe used was an oligonucleotide complementary to all bacterial 16S rRNA so far sequenced. On the basis of these results and of the other observations so far accumulated, the possible eukaryotic nature of epixenosomes is discussed.

The extrusive apparatus is the most prominent and complex structure of epixenosomes. In the present paper the mechanisms activating its ejecting process were investigated by means of in vivo treatments and cytochemical procedures at the ultrastructural level. The results obtained clearly demonstrated that the ejecting process in epixenosomes is triggered by the detection of external signals through membrane receptors and the consequent activation of the adenylate cyclase-cyclic AMP system as a transduction mechanism. The membrane receptors coming into play have an affinity for soybean agglutinin and have a precise localization at the top of the organism, just where a membrane interruption appears as a first step in the whole process. The factors that trigger ejection in nature are still unknown. In the laboratory, ejection was obtained in the presence of adrenalin, which has been proved to bind to the same receptors shown to have affinity for soybean agglutinin. So epixenosomes appear to possess specific binding molecules for a mammalian hormone in the appropriate location, i.e., in the plasma membrane, and this hormone induces a precise biological response. These results are particularly interesting if we consider that epixenosomes are enigmatic organisms in which prokaryotic and eukaryotic characteristics appear to coexist.

As already reported, epixenosomes have a complex cell organization although they lack membranous organelles and a nuclear envelope. Different morphological compartments have been evidenced: (1) a space (IM) between the 2 external membranes in which acid phosphatase has been observed; (2) an apical, electrondense zone (DZ) not delimited by a membrane or thin envelope, containing DNA and basic proteins; (3) a roundish body (RB), 200 nm in diameter, containing reserve polysaccharides and enzymes, in particular, peroxidase; (4) an extrusive apparatus (EA) consisting of a ribbon with smooth surfaces in which lectin binding sites are present, and immersed in a complex matrix different from the remaining cytoplasm. Along its internal and external limits, active adenylate cyclase has been evidenced. At its top, a 'ring' of peroxidase is present; (5) a basket surrounding the extrusive apparatus probably consisting of tubulin microtubules. The cytochemical data, especially the precise localization of the enzymes shown here, indicate that in epixenosomes, a functional compartmentalization corresponds to a structural complexity, in spite of the absence of internal true membranes.

Summary In the present study the variations of the epixenosomal band and the behavior of epixenosomes during the divisional morphogenesis of Euplotodium itoi have been analyzed. From the results obtained it arises that the multiplication, the precise redistribution between the two daughter E. itoi cells, and the transformation of epixenosomes from form I to form II are all processes well coordinated with the reproductive cycle of the ciliate. Euplotidium can live and divide even without epixenosomes. It appears, however, that not only E. itoi has a strong influence on epixenosomes but that the epixenosomes themselves also influence the ciliate host in some way.

We combined morphological (i.e. live, stained, scanning, and transmission electron microscopy) with morphometric and molecular analysis to describe a ciliate species collected from shallow reefs in Guam, grown, and maintained in our laboratory. The species was recognized as a member of Euplotidium, and compared with established species of the genus: Euplotidium itoi Ito 1958; Euplotidium psammophilus (Vacelet 1961) Borror 1972; Euplotidium arenarium Magagnini and Nobili 1964; Euplotidium helgae Hartwig 1980; Euplotidium prosaltans Tuffrau 1985, and Euplotidium smalli Lei, Choi and Xu, 2002. To obtain more elements to compare the species, new morphometric data and additional SSU rRNA gene sequences of E. itoi and of E. arenarium are reported. On the basis of this comparison, we established the new species Euplotidium rosati that has a cirral pattern composed of 12 frontoventral and six transverse cirri, and lacks the left marginal cirrus. Euplotidium rosati harbors on its dorsal surface epixenosomes, the peculiar extrusive symbionts described in other Euplotidium species. The whole body of our observations together with the analysis of the data available in the literature leads us to propose a redefinition of the genus. The results may also be useful to clarify the tangled relationship between Euplotidium and Gastrocirrhus.

Summary A characteristic feature of Euplotidium itoi is the cortical band which containsepixenosomes. In some specimens the formation of deep cortical invaginations has been observed in the cortical band. These invaginations contain foreign bacteria and a few epixenosomes. Successively, intracellular vacuoles are formed, delimited by the whole cortex with the plasmamembrane inward and the alveolar system toward the ciliate cytoplasm. No signs of digestion could be observed within these vacuoles. The alveolar system is gradually dissolved in small regions. Very likely, bacteria and epixenosomes escape from the vacuole at these sites which are delimited by a single unit membrane only. Once in the Euplotidium cytoplasm bacteria are surrounded by rough endoplasmic reticulum membranes. The significance of this process, quite unusual in ciliates, is far from being understood; it is not even clear whether epixenosomes have a role in it and why it takes place only at the level of the epixenosomal band. Moreover is not known what happens to the episymbionts once introduced inside the host cell. As far as bacteria are concerned, the constant alterations of mitochondrial features found in specimens with cortical vacuoles, suggest an infectious nature.

Epixenosomes, epibiontic organisms living on the protozoon ciliate Euplotidium itoi, like prokaryotes, lack a true nucleus surrounded by a nuclear envelope. However, their cytoplasm, at least in the mature state, is somehow compartmentalized by the presence of a complex extrusive apparatus surrounded by a form of basket consisting of bundles of tubules (BT), 20–24 nm in diameter, delineated by a wall made of globular subunits. The nature of these tubules was investigated by means of in vivo experiments, ultrastructural analysis and immunocytochemistry by both fluorescence and electron microscopy. It has been found that BT, besides being well preserved when fixed in the presence of tannic acid, are sensitive to factors known to depolymerize cytoplasmic microtubules in a variety of cells such as nocodazole and cold and show a positive immunoreaction against different antitubulin antibodies. On the basis of all the results obtained, the hypothesis that the epixenosomal BT consist of tubulin acquires a strong credibility. This is particularly interesting if we consider that tubulin in prokaryotes has never been conclusively demonstrated.

Analyse ultrastructurale par microscopie electronique a transmission et a balayage de la bande de particules limitant le corps cellulaire de E. itoi

The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis' 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes' making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such studies confirm, refine, or replace our hypothesis, they should nevertheless further our understanding of the origin of eukaryotic cells.

Epixenosomes, ectosymbionts on hypotrich ciliates (genus Euplotidium ) defend their host against the ciliate predator Litonotus lamella. Although here only Euplotidium itoi and Euplotidium arenarium from tide pools along a rocky shore near Leghorn (Ligurian sea) were studied in detail, these epibionts are certainly present on specimens of E. itoi and on other Euplotidium species in similar north coastal habitats. The complex life history of epixenosomes has two main stages. In stage I, cells with typical prokaryotic structure divide by binary fission. Stage II cells show complex organization with different cytoplasmic compartments where an extrusive apparatus within a proteinaceous matrix, although not membrane-bounded, differs from the remaining cytoplasm. The ejection process is involved in defense; extrusive apparatus is surrounded by a basket consisting of bundles of tubules. These tubules, 22 ± 3 nm in diameter, delimited by a wall made up of globular structures, are sensitive to inhibitor of tubulin polymerization (nocodazole/4°C temperature) and react positively with different antitubulin antibodies, two of which are monoclonal. The prokaryotic vs. eukaryotic nature of epixenosomes was resolved by comparative sequence analysis of amplified small subunit rRNA genes and in situ hybridization with fluorescently labeled rRNA-targeted polynucleotide probes. These unique ectosymbionts are phylogenetically related to Verrucomicrobia . Epixenosomes represent marine symbionts in this recently discovered division of the Bacteria.