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Ice-binding proteins (IBPs) bind to ice crystals and control their growth, enabling host organisms to adapt to subzero temperatures. By binding to ice, IBPs can affect the shape and recrystallization of ice crystals. The shapes of ice crystals produced by IBPs vary and are partially due to which ice planes the IBPs are bound to. Previously, we have described a bacterial IBP found in the metagenome of the symbionts of Euplotes focardii ( EfcIBP). EfcIBP shows remarkable ice recrystallization inhibition activity. As recrystallization inhibition of IBPs and other materials are important to the cryopreservation of cells and tissues, we speculate that the EfcIBP can play a future role as an ice recrystallization inhibitor in cryopreservation applications. Here we show that EfcIBP results in a Saturn-shaped ice burst pattern, which may be due to the unique ice-plane affinity of the protein that we elucidated using the fluorescent-based ice-plane affinity analysis. EfcIBP binds to ice at a speed similar to that of other moderate IBPs (5 ± 2 mM-1 s-1); however, it is unique in that it binds to the basal and previously unobserved pyramidal near-basal planes, while other moderate IBPs typically bind to the prism and pyramidal planes and not basal or near-basal planes. These insights into EfcIBP allow a better understanding of the recrystallization inhibition for this unique protein

We announce here the draft genome sequence of a new Pseudomonas strain, named Pseudomonas sp. strain ef1, associated with the cold-adapted Antarctic ciliate Euplotes focardii. The genome sequence is 6,228,167 bp long with a G+C content of 59.7%.

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.

From the ciliate Euplotes focardii, collected from Ross Sea coastal waters, Antarctica, a new-skeleton diterpenoid, epoxyfocardin, was isolated as a 85:15 mixture of hemiacetals 8a and 8b. The absolute configuration of 8a/8b was determined from Mosher's esters 11a/11b and 12a/12b. Focardin 9a/9b, most likely a biogenetic precursor of 8a/8b, was also isolated as a minor component. Focardin, and particularly epoxyfocardin, proved to be toxic towards representatives of ciliate communities from Antarctic, temperate, tropical, and equatorial environments, constituting the first example of ecologically relevant metabolites from ciliate species that inhabit polar ecosystems.

Summary: Microtubules are the major cytoskeletal component in Euplotes focardii . The spatial organization of the microtubular system of this marine antarctic ciliate is described. Three different methods were employed: immunofluorescence using antitubulin antibodies, scanning electron microscopy, and transmission electron microscopy. The general arrangement of the cytoskeletal microtubular system is similar to that described for temperate species of Euplotes . Basal bodies, microtubular derivatives associated with the kinetosomes and superficial microtubular networks in both dorsal and ventral sides are stable at the low temperatures this species must bear. Slight differences are reported in the disposition of the microtubules on the somatic cortex, As for the oral cytoskeleton the left side infraciliature is reinforced by a complex microtubular system: on the one hand, a developed microtubular network spreading under and among the paramembranelles, and on the other hand microtubules reinforcing the oral crests.

The activity of citrate synthase (CS), a mitochondrial enzyme in the energy metabolism pathway, was studied in Euplotes focardii (Hypotrichia) and Uronema marinum (Scuticociliatia), isolated from samples of Antarctic seawater and, for comparison in E. vannus and U. nigricans, related ciliates from the Adriatic. The temperature-dependent characteristics of CS were investigated in the range 0–55°C, to evaluate mechanisms of thermal regulation, such as optimal temperature levels, activation energy, and kinetic parameters. CS activity was up to double that recorded in temperate organisms at reaction temperatures between 0 and 10°C. The optimal temperature for enzymatic activity was independent of T amb. Activation energy for CS was significantly lower in Antarctic ciliates than in temperate ones, indicating a significant increase in the reaction rate. K M at various temperatures of the reaction substrates indicated the higher affinity of CS for acetyl-CoA in both Antarctic organisms at lower temperatures. The data for thermal profiles for K M, showing greater differences between E. focardii and E. vannus than between U. marinum and U. nigricans, support the distinctive physiological characteristics of each species. E. focardii is an endemic and psychrophilic species, whereas U. marinum is a typically ubiquitous species, more adaptable to environmental changes.

A ciliate, Euplotes focardii, endemic in low‐bottom sea waters of Antarctic coasts was shown to possess heat‐shock genes of the universally conserved family hsp70 that remain trascriptionally inert in heat‐shocked cells. Yet these genes appeared to be represented by thousands of copies in the functional genome that is carried in the cell somatic (macro)nucleus, and constitutively expressed in cells growing under standard temperature conditions in a cold room. It was suggested that these findings reflect a long evolutionary existence that E. focardii has spent in an environment unique for its permanent thermal stability at subzero temperature.

Oxidative stress is a particularly severe threat to Antarctic marine polar organisms because they are exposed to high dissolved oxygen and to intense UV radiation. This paper reports the features of three superoxide dismutases from the Antarctic psychrophilic ciliate Euplotes focardii that faces two environmental challenges, oxidative stress and low temperature. Two out of these are Cu,Zn superoxide dismutases (named Ef-SOD1a and Ef-SOD1b) and one belongs to the Mn-containing group (Ef-SOD2). Ef-SOD1s and Ef-SOD2 differ in their evolutionary history, expression and overall structural features. Ef-SOD1 genes are expressed at different levels, with Ef-SOD1b mRNA 20-fold higher at the ciliate optimal temperature of growth (4 °C). All Ef-SOD enzymes are active at 4 °C, consistent with the definition of cold-adapted enzymes. At the same time, they display temperatures of melting in the range 50–70 °C and retain residual activity after incubation at 65–75 °C. Supported by data of molecular dynamics simulation, we conclude that the E. focardii SODs combine cold activity, local molecular flexibility and thermo tolerance.