The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.

Horizontal gene transfer and silver nanoparticles production in a new Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii.

We isolated a novel bacterial strain from a prokaryotic consortium associated to the psychrophilic marine ciliate Euplotes focardii, endemic of the Antarctic coastal seawater. The 16S rDNA sequencing and the phylogenetic analysis revealed the close evolutionary relationship to the Antarctic marine bacterium Marinomonas sp. BSw10506 and the sub antarctic Marinomonas polaris. We named this new strain Marinomonas sp. ef1. The optimal growth temperature in LB medium was 22 °C. Whole genome sequencing and analysis showed a reduced gene loss limited to regions encoding for transposases. Additionally, five genomic islands, e.g. DNA fragments that facilitate horizontal gene transfer phenomena, were identified. Two open reading frames predicted from the genomic islands coded for enzymes belonging to the Nitro-FMN-reductase superfamily. One of these, the putative NAD(P)H nitroreductase YfkO, has been reported to be involved in the bioreduction of silver (Ag) ions and the production of silver nanoparticles (AgNPs). After the Marinomonas sp. ef1 biomass incubation with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. The AgNPs were relatively small in size (50 nm) and had a strong antimicrobial activity against twelve common nosocomial pathogenic microorganisms including Staphylococcus aureus and two Candida strains. To our knowledge, this is the first report of AgNPs biosynthesis by a Marinomonas strain. This biosynthesis may play a dual role in detoxification from silver nitrate and protection from pathogens for the bacterium and potentially for the associated ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common pathogens.

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.

Unifying ecological stoichiometry and metabolic theory to predict production and trophic transfer in a marine planktonic food web.

Two ecological frameworks have been used to explain multitrophic interactions, but rarely in combination: (i) ecological stoichiometry (ES), explaining consumption rates in response to consumers' demand and prey's nutrient content; and (ii) metabolic theory of ecology (MTE), proposing that temperature and body mass affect metabolic rates, growth and consumption rates. Here we combined both, ES and MTE to investigate interactive effects of phytoplankton prey stoichiometry, temperature and zooplankton consumer body mass on consumer grazing rates and production in a microcosm experiment. A simple model integrating parameters from both frameworks was used to predict interactive effects of temperature and nutrient conditions on consumer performance. Overall, model predictions reflected experimental patterns well: consumer grazing rates and production increased with temperature, as could be expected based on MTE. With decreasing algal food quality, grazing rates increased due to compensatory feeding, while consumer growth rates and final biovolume decreased. Nutrient effects on consumer biovolume increased with increasing temperature, while nutrient effects on grazing rates decreased. Highly interactive effects of temperature and nutrient supply indicate that combining the frameworks of ES and MTE is highly important to enhance our ability to predict ecosystem functioning in the context of global change.

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.

UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

Effects of elevated temperature on the toxicity of copper and oxytetracycline in the marine model, Euplotes crassus: a climate change perspective.

Trace metals and broad-spectrum antibiotic drugs are common environmental contaminants, the importance of which is increasing due to global climate change-related effects. In the present study, the biological model organism E. crassus was first acclimated to five temperatures, from 25 °C to 33 °C, followed by exposure to nominal concentrations of copper, the antibiotic model compound oxytetracycline and mixtures of both, at increasing thermal conditions. Variations of temperature-related toxicity were assessed by two high-level endpoint tests, survival and replication rates, and two sublethal parameters: endocytosis rate and lysosomal membrane stability. The selected toxicants presented opposite behaviours as the protozoa's survival rates increased following an increasing thermal gradient in the oxytetracycline-related treatments, and a decline of tolerance in metal-related treatments was observed. Results of tests combining binary mixtures of tested toxicants showed a complex pattern of responses.

Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus.

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4-5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases. By analyzing both esterase and phospholipase A2 activity, we determined the thermostability and the optimal pH, temperature dependence and substrates of these enzymes. We demonstrated that EfPLP shows the characteristics of a psychrophilic phospholipase. Furthermore, we analyzed the enzymatic activity of three engineered versions of the EfPLP, in which unique residues of EfPLP, Gly80, Ala201 and Val204, were substituted through site-directed mutagenesis with residues found in the E. crassus homolog (Glu, Pro and Ile, respectively). Additionally, three corresponding mutants of EcPLP were also generated and characterized. These analyses showed that the substitution of amino acids with rigid and bulky charged/hydrophobic side chain in the psychrophilic EfPLP confers enzymatic properties similar to those of the mesophilic patatin-like phospholipase, and vice versa. This is the first report on the isolation and characterization of a cold-adapted patatin-like phospholipase from eukaryotes. The results reported in this paper support the idea that enzyme thermal-adaptation is based mainly on some amino acid residues that influence the structural flexibility of polypeptides and that EfPLP is an attractive biocatalyst for industrial processes at low temperatures.

Morphological, physiological, and molecular evidences suggest that Euplotes parawoodruffi is a junior synonym of Euplotes woodruffi (Ciliophora, Euplotida).

Euplotes woodruffi Gaw 1939 is a striking ciliate species commonly used in many fields of protistology. Euplotes parawoodruffi Song and Bradbury 1997 is separated from E. woodruffi by several morphological differences (e.g. the absence of pre-oral pouch) and in particular the brackish habitat (vs. freshwater). However, recent molecular data dispute the separation of E. woodruffi into two species. Based on a brackish isolate from the southeast of China, we investigated the morphology, molecular phylogeny, and adaptation of E. woodruffi using living observation, protargol impregnation, gene sequencing, and cultivation experiments. The morphological variability present in our isolate encompasses the previous descriptions of E. woodruffi and E. parawoodruffi. The pre-oral pouch, which occurs in living cells of freshwater isolates, is also recognizable in the brackish specimens. Laboratory experiments suggested that the brackish isolate could acclimatize and reproduce in both brackish and fresh water. The molecular analyses using 13 available small subunit (SSU) rRNA gene sequences showed that the minimum pairwise sequence similarity is as high as 99.2% and nine sequences from both brackish and freshwater isolates are even 100% matched. Therefore, we conclude that E. parawoodruffi is a junior synonym of E. woodruffi.

Structural thermal adaptation of ß-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii.

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three ß-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the ß-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii ß-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic ß-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.

Effects of connectivity and recurrent local disturbances on community structure and population density in experimental metacommunities.

Metacommunity theory poses that the occurrence and abundance of species is a product of local factors, including disturbance, and regional factors, like dispersal among patches. While metacommunity ideas have been broadly tested there is relatively little work on metacommunities subject to disturbance. We focused on how localized disturbance and dispersal interact to determine species composition in metacommunities. Experiments conducted in simple two-patch habitats containing eight protozoa and rotifer species tested how dispersal altered community composition in both communities that were disturbed and communities that connected to refuge communities not subject to disturbance. While disturbance lowered population densities, in disturbed patches connected to undisturbed patches this was ameliorated by immigration. Furthermore, species with high dispersal abilities or growth rates showed the fastest post-disturbance recovery in presence of immigration. Connectivity helped to counteract the negative effect of disturbances on local populations, allowing mass-effect-driven dispersal of individuals from undisturbed to disturbed patches. In undisturbed patches, however, local population sizes were not significantly reduced by emigration. The absence of a cost of dispersal for undisturbed source populations is consistent with a lack of complex demography in our system, such as age- or sex-specific emigration. Our approach provides an improved way to separate components of population growth from organisms' movement in post-disturbance recovery of (meta)communities. Further studies are required in a variety of ecosystems to investigate the transient dynamics resulting from disturbance and dispersal.

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating.

Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii, based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the three-dimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.

Phase locking, the Moran effect and distance decay of synchrony: experimental tests in a model system.

Spatially separated populations of many species fluctuate synchronously. Synchrony typically decays with increasing interpopulation distance. Spatial synchrony, and its distance decay, might reflect distance decay of environmental synchrony (the Moran effect), and/or short-distance dispersal. However, short-distance dispersal can synchronize entire metapopulations if within-patch dynamics are cyclic, a phenomenon known as phase locking. We manipulated the presence/absence of short-distance dispersal and spatially decaying environmental synchrony and examined their separate and interactive effects on the synchrony of the protist prey species Tetrahymena pyriformis growing in spatial arrays of patches (laboratory microcosms). The protist predator Euplotes patella consumed Tetrahymena and generated predator-prey cycles. Dispersal increased prey synchrony uniformly over both short and long distances, and did so by entraining the phases of the predator-prey cycles. The Moran effect also increased prey synchrony, but only over short distances where environmental synchrony was strongest, and did so by increasing the synchrony of stochastic fluctuations superimposed on the predator-prey cycle. Our results provide the first experimental demonstration of distance decay of synchrony due to distance decay of the Moran effect. Distance decay of the Moran effect likely explains distance decay of synchrony in many natural systems. Our results also provide an experimental demonstration of long-distance phase locking, and explain why cyclic populations provide many of the most dramatic examples of long-distance spatial synchrony in nature.

Stronger inducible defences enhance persistence of intraguild prey.

1. Intraguild predation is widespread in nature despite its potentially destabilizing effect on food web dynamics. 2. Anti-predator inducible defences affect both birth and death rates of populations and have the potential to substantially modify food web dynamics and possibly increase persistence of intraguild prey. 3. In a chemostat experiment, we investigated the long-term effects of inducible defences on the dynamics of aquatic microbial food webs consisting of an intraguild predator, intraguild prey, and a basal resource. We controlled environmental conditions and selected strains of intraguild prey that varied in the strength of expressed inducible defences. 4. We found that intraguild prey with a stronger tendency to induce an anti-predator morphology persist for significantly longer periods of time. In addition, model selection analysis implied that flexibility in defensive phenotype (inducibility itself) is most likely the factor responsible for the enhanced persistence. 5. As patterns at the community level often emerge as a result of the life-history traits of individuals, we propose that inducible defences increase the persistence of populations and may contribute to the widespread occurrence of theoretically unstable intraguild predation systems in nature.

A novel protein kinase from the ciliate Euplotes raikovi with close structural identity to the mammalian intestinal and male-germ cell kinases: characterization and functional implications in the autocrine pheromone signaling loop.

In the free-living ciliate Euplotes raikovi, we identified (and designated as Er-MAPK1) a protein kinase of 631 amino acids, that appears to be constantly phosphorylated in cells which are in growth stage and interact in autocrine fashion with their water-soluble signal pheromones. Er-MAPK1 is specified by a gene that requires a+1 translational frame-shift to be expressed. Its amino-terminal region represents a canonical catalytic domain and carries an activation loop distinctive of the mitogen-activated protein kinases, with the Thr-Asp-Tyr motif deputed to be site of double phosphorylation. In contrast, the carboxy-terminal region appears to be structurally unique. It shows a strongly basic amino acid composition, is very rich in glycine repetitions, and contains a bipartite signal for translocation of Er-MAPK1 into the nucleus.

Growth phase and elemental stoichiometry of bacterial prey influences ciliate grazing selectivity.

Protozoa are known to selectively graze bacteria and can differentiate prey based on size and viability, but less is known about the effects of prey cellular composition on predator selectivity. We measured the effect of growth phase and elemental stoichiometry of Escherichia coli on grazing by two ciliates, Euplotes vannus and Cyclidium glaucoma. Bacterial cells of a single strain were transformed with green and red fluorescent protein and harvested from culture at differing growth stages. Cells in exponential growth phase had low carbon:phosphorus (39) and nitrogen:phosphorus (9) ratios, while cells from stationary phase had high carbon:phosphorus of 104 and nitrogen:phosphorus of 26. When offered an equal mixture of both types of bacteria, Cyclidium grazed stationary phase, high carbon:phosphorus, high nitrogen:phosphorus cells to 22% of initial abundance within 135 min, while Euplotes reduced these cells to 33%. Neither ciliate species decreased the abundance of the exponential phase cells, lower carbon:phosphorus and nitrogen:phosphorus, relative to control treatments. Because protozoa have higher nitrogen:phosphorus and carbon:phosphorus ratios than their prokaryotic prey, this study raises the possibility that it may be advantageous for protozoa to preferentially consume more slowly growing bacteria.

Phase-locking and environmental fluctuations generate synchrony in a predator-prey community.

Spatially synchronized fluctuations in system state are common in physical and biological systems ranging from individual atoms to species as diverse as viruses, insects and mammals. Although the causal factors are well known for many synchronized phenomena, several processes concurrently have an impact on spatial synchrony of species, making their separate effects and interactions difficult to quantify. Here we develop a general stochastic model of predator-prey spatial dynamics to predict the outcome of a laboratory microcosm experiment testing for interactions among all known synchronizing factors: (1) dispersal of individuals between populations; (2) spatially synchronous fluctuations in exogenous environmental factors (the Moran effect); and (3) interactions with other species (for example, predators) that are themselves spatially synchronized. The Moran effect synchronized populations of the ciliate protist Tetrahymena pyriformis; however, dispersal only synchronized prey populations in the presence of the predator Euplotes patella. Both model and data indicate that synchrony depends on cyclic dynamics generated by the predator. Dispersal, but not the Moran effect, 'phase-locks' cycles, which otherwise become 'decoherent' and drift out of phase. In the absence of cycles, phase-locking is not possible and the synchronizing effect of dispersal is negligible. Interspecific interactions determine population synchrony, not by providing an additional source of synchronized fluctuations, but by altering population dynamics and thereby enhancing the action of dispersal. Our results are robust to wide variation in model parameters representative of many natural predator-prey or host-pathogen systems. This explains why cyclic systems provide many of the most dramatic examples of spatial synchrony in nature.

Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii.

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.

[How translation termination factor eRF1 Euplotes does not recognise UGA stop codon].

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.

Ribosomal cold-adaptation: characterization of the genes encoding the acidic ribosomal P0 and P2 proteins from the Antarctic ciliate Euplotes focardii.

Molecular adaptation at low temperature requires specificities represented mainly by modifications in the gene sequence and consequently in the protein primary structure. To characterize the molecular mechanisms responsible for ribosome cold-adaptation, we compared the ribosomal P0 and P2 genes from the Antarctic ciliate Euplotes focardii with homologous genes from mesophilic organisms, including the ciliates Tetrahymena thermophila and non cold-adapted Euplotes species. This analysis revealed the presence of non synonymous mutations unique to E. focardii. In the P0 protein the mutations produced amino acid substitutions that increased the molecular flexibility that may facilitate a conformational adjustment associated with the interaction with the GTPase center of the large subunit rRNA, and increased the hydrophobicity of the region involved in the interaction with P1/P2 heterodimer, probably to keep associated the ribosomal stalk in the cold. In the P2 protein the mutations produced amino acid substitutions that increased the N-terminus flexibility, which may facilitate interactions with P1 protein in the formation of the heterodimer, and reduced the mobility of the C-terminus, to stabilize the stalk during ribosomal activity. Finally, P proteins appeared to be valid markers for investigating the phylogenetic origin of early eukaryotes.