Search

In the present review, we summarize genome mining of genomic data obtained from the psychrophilic Antarctic marine ciliate Euplotes focardii and its evolutionary-close mesophilic cosmopolitan counterpart E. crassus . This analysis highlights adaptation strategies that are unique to the Antarctic ciliate, including antioxidant gene duplication and distinctive substitutions that may play roles in increased drug binding affinity and enzyme reaction rate in cold environments. Enzymes from psychrophiles are usually characterized by high activities and reaction rates at low temperatures compared with their counterparts from mesophiles and thermophiles. As a rule, catalyst cold activity derives from an increased structural flexibility that may lead to protein denaturation in response to temperature fluctuation. Molecular thermolability has been a major drawback of using macromolecules from psychrophiles in industrial applications. Here, we report a case study in which the role of peculiar amino acid substitution in cold adaptation is demonstrated by site-directed mutagenesis. Combined with a rational design approach, these substitutions can be used for site-directed mutagenesis to obtain cold-active catalysts that are structurally stable. Furthermore, molecular docking analysis of β-tubulin isotypes extrapolated from E. focardii and E. crassus genomes allowed us to obtain additional insight on the taxol binding site and drug affinity. E. focardii genome mining and the comparison with the mesophilic sibling counterpart can be used as an inspiration for molecular engineering for medical and industrial applications.

Marine microorganisms have been demonstrated to be an important source for bioactive molecules. In this paper we report the synthesis of Ni nanoparticles (NiSNPs) used as reducing and capping agents for five bacterial strains isolated from an Antarctic marine consortium: Marinomonas sp. ef1, Rhodococcus sp. ef1, Pseudomonas sp. ef1, Brevundimonas sp. ef1, and Bacillus sp. ef1. The NiSNPs were characterized by Ultraviolet-visible (UV-vis) spectroscopy, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) spectroscopic analysis. The maximum absorbances in the UV-Vis spectra were in the range of 374 nm to 422 nm, corresponding to the Surface plasmon resonance (SPR) of Nickel. DLS revealed NiSNPs with sizes between 40 and 45 nm. All NiSNPs were polycrystalline with a face-centered cubic lattice, as revealed by XRD analyses. The NiSNPs zeta potential values were highly negative. TEM analysis showed that the NiSNPs were either spherical or rod shaped, well segregated, and with a size between 20 and 50 nm. The FTIR spectra revealed peaks of amino acid and protein binding to the NiSNPs. Finally, all the NiSNPs possess significant antimicrobial activity, which may play an important role in the management of infectious diseases affecting human health.

In this study, we report on the synthesis of silver nanoparticles (AgNPs) achieved by using three bacterial strains Rhodococcus , Brevundimonas and Bacillus as reducing and capping agents, newly isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii . After incubation of these bacteria with a 1 mM solution of AgNO 3 at 22 °C, AgNPs were synthesized within 24 h. Unlike Rhodococcus and Bacillus , the reduction of Ag + from AgNO 3 into Ag 0 has never been reported for a Brevundimonas strain. The maximum absorbances of these AgNPs in the UV-Vis spectra were in the range of 404 nm and 406 nm. EDAX spectra showed strong signals from the Ag atom and medium signals from C, N and O due to capping protein emissions. TEM analysis showed that the NPs were spherical and rod-shaped, with sizes in the range of 20 to 50 nm, and they were clustered, even though not in contact with one another. Besides aggregation, all the AgNPs showed significant antimicrobial activity. This biosynthesis may play a dual role: detoxification of AgNO 3 and pathogen protection against both the bacterium and ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common nosocomial pathogens.

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., (© 2021. The Author(s).)

In the last decade, metal nanoparticles (NPs) have gained significant interest in the field of biotechnology due to their unique physiochemical properties and potential uses in a wide range of applications. Metal NP synthesis using microorganisms has emerged as an eco-friendly, clean, and viable strategy alternative to chemical and physical approaches. Herein, an original and efficient route for the microbial synthesis of copper NPs using bacterial strains newly isolated from an Antarctic consortium is described. UV-visible spectra of the NPs showed a maximum absorbance in the range of 380-385 nm. Transmission electron microscopy analysis showed that these NPs are all monodispersed, spherical in nature, and well segregated without any agglomeration and with an average size of 30 nm. X-ray powder diffraction showed a polycrystalline nature and face centered cubic lattice and revealed characteristic diffraction peaks indicating the formation of CuONPs. Fourier-transform infrared spectra confirmed the presence of capping proteins on the NP surface that act as stabilizers. All CuONPs manifested antimicrobial activity against various types of Gram-negative; Gram-positive bacteria; and fungi pathogen microorganisms including Escherichia coli , Staphylococcus aureus , and Candida albicans . The cost-effective and eco-friendly biosynthesis of these CuONPs make them particularly attractive in several application from nanotechnology to biomedical science.

The synthesis of silver nanoparticles (AgNPs) by microorganisms recently gained a greater interest due to its potential to produce them in various sizes and morphologies. In this study, for AgNP biosynthesis, we used a new Pseudomonas strain isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii . After incubation of Pseudomonas cultures with 1 mM of AgNO 3 at 22 °C, we obtained AgNPs within 24 h. Scanning electron (SEM) and transmission electron microscopy (TEM) revealed spherical polydispersed AgNPs in the size range of 20-70 nm. The average size was approximately 50 nm. Energy dispersive X-ray spectroscopy (EDS) showed the presence of a high intensity absorption peak at 3 keV, a distinctive property of nanocrystalline silver products. Fourier transform infrared (FTIR) spectroscopy found the presence of a high amount of AgNP-stabilizing proteins and other secondary metabolites. X-ray diffraction (XRD) revealed a face-centred cubic (fcc) diffraction spectrum with a crystalline nature. A comparative study between the chemically synthesized and Pseudomonas AgNPs revealed a higher antibacterial activity of the latter against common nosocomial pathogen microorganisms, including Escherichia coli, Staphylococcus aureus and Candida albicans . This study reports an efficient, rapid synthesis of stable AgNPs by a new Pseudomonas strain with high antimicrobial activity.

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.

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., (© 2016 Federation of European Biochemical Societies.)

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI's prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in "transmembrane transporter activity" and "oxidoreductase activity". Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment.

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 β-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 β-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 β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

Tubulin dimers of psychrophilic organisms can polymerize into microtubules at temperatures below 4 degrees C, at which non-cold-adapted microtubules disassemble. This capacity requires specificities in the structure and/or in the posttranslational modifications of the tubulin subunits. A contribution to the knowledge of these specificities was provided by the finding that the amino acid sequence of the alpha-tubulin of the Antarctic ciliate Euplotes focardii contains substitutions that, in addition to conferring an increased hydrophobicity to the molecule, modify sites that are involved in alpha-/alpha-tubulin lateral contacts between protofilaments. At the level of the coding sequence, the alpha-tubulin gene of E. focardii revealed an A+T content appreciably higher than in its homologs in ciliates of temperate waters. This was interpreted as an adaptation to favor DNA strand separation in an environment which is energetically adverse.

Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat-shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii, and behave as psychrophile and psychrotroph micro-organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell's somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 degrees C, a much smaller increase was revealed in heat-shocked cells of E. focardii. These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.

In cold poikilotherm organisms, microtubule assembly is promoted at temperatures below 4 degrees C and cold-induced depolymerization is prevented. On the basis of the results of investigations on cold-adapted fishes, the property of cold adaptation is ascribed to intrinsic characteristics of the tubulins. To fully understand cold adaptation, we studied the tubulins of Euplotes focardii, an Antarctic ciliated protozoan adapted to temperatures ranging from -2 to +4 degrees C. In this organism, we had previously sequenced one beta-tubulin gene and, then identified three other genes (denoted as beta-T1, beta-T2, beta-T3 and beta-T4). Here we report that the amino acid sequence of the carboxy-terminal domain predicted from the beta-T3 gene (apparently the most expressed of the gene family) contains six modifications (five substitutions and one insertion) of conserved residues, unique with respect to all the other known beta-tubulin sequences. These modifications can change the structural conformation of the carboxy-terminal domain. Furthermore, in the variable terminal end of that domain, a consensus sequence for a phosphorylation site is present, and the residue Glu-438, the most frequent site for polyglutamylation in beta-tubulin, is substituted by Asp. Starting from these observations, we showed that in E. focardii only alpha-tubulin is polyglutamylated, while beta-tubulin undergoes phosphorylation. Polyglutamylated microtubules appear to colocalize with cilia and microtubular bundles, all structures in which microtubules undergo a sliding process. This finding supports the idea that alpha-tubulin polyglutamylation is involved in the interaction between tubulin and motor microtubule-associated proteins. Phosphorylation, usually a rare posttranslational modification of beta-tubulin, which is found extensively distributed in the beta-tubulin of this cold-adapted organism, may play a determinant role in the dynamic of polymerization and depolymerization at low temperatures.