Crystal structure of the pheromone Er-13 from the ciliate Euplotes raikovi, with implications for a protein-protein association model in pheromone/receptor interactions.

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation.

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.

N­(6­Aminohexyl)­5­chloro­1­naphthalenesulfonamide, a centrin antagonist, inhibits Tb3+/peptides-binding properties.

N­(6­Aminohexyl)­5­chloro­1­naphthalenesulfonamide (W-7), a kind of adjuvant chemotherapy, can bind to calmodulin and inhibit Ca2+/calmodulin-regulated enzyme activities and cell proliferation. Similar to calmodulin, euplotes octocarinatus centrin (EoCen) belongs to EF-hand superfamily of calcium-binding proteins. It is associated with nucleotide excision repair (NER), cell division cycle and ciliogenesis. In the present study, the comparative interaction of W-7 with EoCen was first examined by using various spectroscopic, calorimetric methods and molecular docking. The obtain results recommend that only one W-7 molecule is identified binding to the C-terminal hydrophobic pocket of centrin that normally plays a role in anchoring targets. Methyl groups of Ala126, Met141, Ile161 and M162 of C-terminal may react with W-7 chloronaphthalene ring, other aliphatic or aromatic side-chains in a deep hydrophobic pocket of protein. Circular dichroism (CD) and fluorescence lifetime experiments reveal that W-7 triggers a conformational change of centrin. As a result, W-7 is identified to be an antagonist of centrin. It appears to inhibit the centrin-mediated activation of target proteins by blocking the hydrophobic pocket. Moreover, the complex formation leads to affinity decrease of Tb3+ binding to C-terminal of protein and self-assembly affected. Our present study provides the first view of centrin recognizing a naphthalene-sulfonamide derivative. It is proposed that W-7 and its analogues can serve as a useful tool for research on the participation of centrin in biological processes and cell biology-related studies.

Antarctic marine ciliates under stress: superoxide dismutases from the psychrophilic Euplotes focardii are cold-active yet heat tolerant enzymes.

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.

Crystal structure of the trimeric N-terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions.

Centrin is a member of the EF-hand superfamily of calcium-binding proteins, a highly conserved eukaryotic protein that binds to Ca2+ . Its self-assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) was determined by using the selenomethionine single-wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca2+ ion-binding sites I and II, protomer A, and C bound one Ca2+ ion, while protomer B bound two Ca2+ ions. A novel binding site III was observed and the Ca2+ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.

Structure of a bacterial ice binding protein with two faces of interaction with ice.

Ice-binding proteins (IBPs) contribute to the survival of many living beings at subzero temperature by controlling the formation and growth of ice crystals. This work investigates the structural basis of the ice-binding properties of EfcIBP, obtained from Antarctic bacteria. EfcIBP is endowed with a unique combination of thermal hysteresis and ice recrystallization inhibition activity. The three-dimensional structure, solved at 0.84 Å resolution, shows that EfcIBP belongs to the IBP-1 fold family, and is organized in a right-handed ß-solenoid with a triangular cross-section that forms three protein surfaces, named A, B, and C faces. However, EfcIBP diverges from other IBP-1 fold proteins in relevant structural features including the lack of a 'capping' region on top of the ß-solenoid, and in the sequence and organization of the regions exposed to ice that, in EfcIBP, reveal the presence of threonine-rich ice-binding motifs. Docking experiments and site-directed mutagenesis pinpoint that EfcIBP binds ice crystals not only via its B face, as common to other IBPs, but also via ice-binding sites on the C face. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 6EIO.

Identification of cyst wall proteins of the hypotrich ciliate Euplotes encysticus using a proteomics approach.

Euplotes encysticus is a species of Hypotrich ciliates, which form cyst wall by secreting the special substances on encounter of adverse environment. It has critical significance to study the component and mechanism underlying resting cyst, during resisting unfavorable conditions in dormancy induction. The present study was aimed to investigate the effects of cyst wall proteins of Euplotes encysticus by using biochemical methods. Therefore, protein extracts were separated by SDSPAGE, identified and analyzed by MALDI-TOF MS and Bioinformatics tools. We detected 42 cyst wall proteins, 26 were functional proteins and 16 proteins consist of unknown function; which is consistent with cyst wall specificity. These results partially revealed the components of resting cyst wall formed after the cells differentiation of Euplotes encysticus. In addition, our data suggested that the function of cyst wall proteins are more likely involved in the mechanical protection, signal transduction, material transport, protein degradation and energy metabolism to survival, with potentially importance implications in the molecular mechanism of eukaryocyte dormancy under stress condition.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

Cryo-protective effect of an ice-binding protein derived from Antarctic bacteria.

Cold environments are populated by organisms able to contravene deleterious effects of low temperature by diverse adaptive strategies, including the production of ice binding proteins (IBPs) that inhibit the growth of ice crystals inside and outside cells. We describe the properties of such a protein (EfcIBP) identified in the metagenome of an Antarctic biological consortium composed of the ciliate Euplotes focardii and psychrophilic non-cultured bacteria. Recombinant EfcIBP can resist freezing without any conformational damage and is moderately heat stable, with a midpoint temperature of 66.4 °C. Tested for its effects on ice, EfcIBP shows an unusual combination of properties not reported in other bacterial IBPs. First, it is one of the best-performing IBPs described to date in the inhibition of ice recrystallization, with effective concentrations in the nanomolar range. Moreover, EfcIBP has thermal hysteresis activity (0.53 °C at 50 µm) and it can stop a crystal from growing when held at a constant temperature within the thermal hysteresis gap. EfcIBP protects purified proteins and bacterial cells from freezing damage when exposed to challenging temperatures. EfcIBP also possesses a potential N-terminal signal sequence for protein transport and a DUF3494 domain that is common to secreted IBPs. These features lead us to hypothesize that the protein is either anchored at the outer cell surface or concentrated around cells to provide survival advantage to the whole cell consortium.

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca2+ and Tb3+) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

Characterization and comparative analysis of psychrophilic and mesophilic alpha-amylases from Euplotes species: a contribution to the understanding of enzyme thermal adaptation.

The eukaryotic α-amylase isolated from the psychrophilic ciliated protozoon Euplotes focardii (EfAmy) was expressed in Escherichia coli and biochemically characterized. Its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcAmy). The comparison of the amino acid composition and the surface residue composition of the two enzymes indicated a preference for tiny residues and the avoidance of charged, aromatic and hydrophobic residues in EfAmy. Our comparative homology modeling study reveals a lack of surface salt bridges, a decreased number of the surface charged residues, decreased hydrogen bonds and bound ions, and a reduction of aromatic-sulfur interactions, cationic-π interactions and disulfide interactions in EfAmy. In contrast, sequence alignment and homology modeling showed five unconserved prolines located on the surface loops of EcAmy. By analyzing amylolytic activity towards soluble starch as the substrate, we determined the temperature and pH dependence, thermostability and kinetic parameters of these two enzymes. We demonstrated that EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at low temperatures and high thermolability. In contrast, the EcAmy showed mesophilic characteristics with the highest activity at moderate temperatures and a more than 2-fold increased half-life at 50°C compared to EfAmy. The kcat and KM values of EfAmy were higher than those of the mesophilic EcAmy at all tested temperatures. Furthermore, both EfAmy and EcAmy showed maximum activities at pH 9 and maintained high activities in the presence of surfactants. These results suggest the potential applications of EfAmy and EcAmy as ingredients in detergents for industrial applications.

The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ß and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

Structure and protein composition of a basal-body scaffold ("cage") in the hypotrich ciliate Euplotes.

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Domain motions of class I release factor induced by binding with class II release factor from Euplotes octocarinatus.

The binding of both factors (eRF1 and eRF3) is essential for fast kinetics of the termination of protein translation. The C-terminal domain of eRF1 is known to interact with the C domain of eRF3. Eo-eRF1b contains two highly conserved tryptophan residues (W-11 and W-373), W-11 located in the Eo-eRF1b N domain and W-373 located in the Eo-eRF1b C domain. Fluorimetry was used to study the interactions of the proteins. When binding with Eo-eRF3Cm6, the emission peak of Eo-eRF1b is blue shifted, while the emission peak of Eo-eRF1bC has no notable change. Our results suggest that the eRF1-eRF3 interaction induces the N and C domain of eRF1b to become closer to each other.

eRF1aMC and Mg(2+) dependent structure switch of GTP binding to eRF3 in Euplotes octocarinatus.

Eukaryotic translation termination is governed by eRF1 and eRF3. eRF1 recognizes the stop codons and then hydrolyzes peptidyl-tRNA. eRF3, which facilitates the termination process, belongs to the GTPase superfamily. In this study, the effect of the MC domain of eRF1a (eRF1aMC) on the GTPase activity of eRF3 was analyzed using fluorescence spectra and high-performance liquid chromatography. The results indicated eRF1aMC promotes the GTPase activity of eRF3, which is similar to the role of eRF1a. Furthermore, the increased affinity of eRF3 for GTP induced by eRF1aMC was dependent on the concentration of Mg(2+). Changes in the secondary structure of eRF3C after binding GTP/GDP were detected by CD spectroscopy. The results revealed changes of conformation during formation of the eRF3C·GTP complex that were detected in the presence of eRF1a or eRF1aMC. The conformations of the eRF3C·eRF1a·GTP and eRF3C·eRF1aMC·GTP complexes were further altered upon the addition of Mg(2+). By contrast, there was no change in the conformation of GTP bound to free eRF3C or the eRF3C·eRF1aN complex. These results suggest that alterations in the conformation of GTP bound to eRF3 is dependent on eRF1a and Mg(2+), whereas the MC domain of eRF1a is responsible for the change in the conformation of GTP bound to eRF3 in Euplotes octocarinatus.

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.

The interaction between lanthanide (III) and N-terminal domain of Euplotes octocarinatus centrin.

Centrin, a member of calcium-binding proteins, is an essential component for microtubule-organizing center (MTOC). Lanthanide (Ln) ions can increase amounts, enhance stability and orderliness of microtubules which is an important component of cytoskeleton. To investigate the structural basis of the effect of Ln ions on orderliness of microtubules, we focused on the interactions between the isolated N-terminal domain of Euplotes centrin (N-EoCen) and Ln by some combined biophysical and biochemical methods. Our results suggest that Ln ions may bind to the canonical calcium binding sites on N-EoCen. Taking advantage of ligand competition, we first determined the metal-binding affinities of Nd(3+), Eu(3+), Gd(3+) and Tm(3+) with N-EoCen. Major changes of N-EoCen in secondary and tertiary structure are noted while Ln ions bind with N-EoCen through CD spectra and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) fluorescence. N-EoCen exists in the form of monomer and dimer in the presence of Ln ions. These results can provide some insights into the structural basis of how Ln ions achieve biological effect in cell through the centrin protein.

The binding sites of class I release factor (eRF1) toward class II release factor (eRF3) in Euplotes octocarinatus.

The C domain of eRF1 interacts with the C domain of eRF3, and the binding of both factors is essential for fast kinetics of the termination of protein translation. Analysis by computational simulation demonstrated that several peptides involved in Eo-eRF1/Eo-eRF3 interaction directly. Among these peptides, the two motifs GVEDT and GFGG were highly conserved, while the fragment aa338-346 of Eo-eRF1a/b was variable. In additional, I290 and D293 of Eo-eRF1 were also highly conserved. By the site-directed mutagenesis and pull-down analysis, the amino acid D293 in Eo-eRF1bC domain was conformed playing an important role in eRF1-eRF3 interaction. Eo-eRF1a and Eo-eRF1b may select different manners to interact with Eo-eRF3. These studies contribute to the better understanding the mode of eRF1-eRF3 interaction.

Structures, biological activities and phylogenetic relationships of terpenoids from marine ciliates of the genus Euplotes.

In the last two decades, large scale axenic cell cultures of the marine species comprising the family Euplotidae have resulted in the isolation of several new classes of terpenoids with unprecedented carbon skeletons including the (i) euplotins, highly strained acetylated sesquiterpene hemiacetals; (ii) raikovenals, built on the bicyclo[3.2.0]heptane ring system; (iii) rarisetenolides and focardins containing an octahydroazulene moiety; and (iv) vannusals, with a unique C30 backbone. Their complex structures have been elucidated through a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, molecular mechanics and quantum chemical calculations. Despite the limited number of biosynthetic experiments having been performed, the large diversity of ciliate terpenoids has facilitated the proposal of biosynthetic pathways whereby they are produced from classical linear precursors. Herein, the similarities and differences emerging from the comparison of the classical chemotaxonomy approach based on secondary metabolites, with species phylogenesis based on genetic descriptors (SSU-rDNA), will be discussed. Results on the interesting ecological and biological properties of ciliate terpenoids are also reported.