The Influence of Phenol on the Growth, Morphology and Cell Division of Euglena gracilis.

Phenol, a monocyclic aromatic hydrocarbon with various commercial uses, is a major pollutant in industrial wastewater. Euglena gracilis is a unicellular freshwater flagellate possessing secondary chloroplasts of green algal origin. This protist has been widely used for monitoring the biological effect of various inorganic and organic environmental pollutants, including aromatic hydrocarbons. In this study, we evaluate the influence of different phenol concentrations (3.39 mM, 3.81 mM, 4.23 mM, 4.65 mM, 5.07 mM, 5.49 mM and 5.91 mM) on the growth, morphology and cell division of E. gracilis. The cell count continually decreases (p < 0.05-0.001) over time with increasing phenol concentration. While phenol treatment does not induce bleaching (permanent loss of photosynthesis), the morphological changes caused by phenol include the formation of spherical (p < 0.01-0.001), hypertrophied (p < 0.05) and monster cells (p < 0.01) and lipofuscin bodies. Phenol also induces an atypical form of cell division of E. gracilis, simultaneously producing more than 2 (3-12) viable cells from a single cell. Such atypically dividing cells have a symmetric "star"-like shape. The percentage of atypically dividing cells increases (p < 0.05) with increasing phenol concentration. Our findings suggest that E. gracilis can be used as bioindicator of phenol contamination in freshwater habitats and wastewater.

Versatile biotechnological applications of Euglena gracilis.

Euglena gracilis is a freshwater protist possessing secondary chloroplasts of green algal origin. Various physical factors (e.g. UV) and chemical compounds (e.g. antibiotics) cause the bleaching of E. gracilis cells-the loss of plastid genes leading to the permanent inability to photosynthesize. Bleaching can be prevented by antimutagens (i.e. lignin, vitamin C and selenium). Besides screening the mutagenic and antimutagenic activity of chemicals, E. gracilis is also a suitable model for studying the biological effects of many organic pollutants. Due to its capability of heavy metal sequestration, it can be used for bioremediation. E. gracilis has been successfully transformed, offering the possibility of genetic modifications for synthesizing compounds of biotechnological interest. The novel design of the "next generation" transgenic expression cassettes with respect to the specificities of euglenid gene expression is proposed. Moreover, E. gracilis is a natural source of commercially relevant bioproducts such as (pro)vitamins, wax esters, polyunsaturated fatty acids and paramylon (ß-1,3-glucan). One of the highest limitations of large-scale cultivation of E. gracilis is its disability to synthesize essential vitamins B1 and B12. This disadvantage can be overcome by co-cultivation of E. gracilis with other microorganisms, which can synthesize sufficient amounts of these vitamins. Such co-cultures can be used for the effective accumulation and harvesting of Euglena biomass by bioflocculation.

Calpains in cyanobacteria and the origin of calpains.

Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena, Aphanizomenon, Calothrix, Chamaesiphon, Fischerella, Microcystis, Scytonema and Trichormus. Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: (1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively (2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes.

Euglena gracilis can grow in the mixed culture containing Cladosporium westerdijkiae, Lysinibacillus boronitolerans and Pseudobacillus badius without the addition of vitamins B1 and B12.

Euglena gracilis is a freshwater flagellate possessing secondary chloroplast of green algal origin. This protist has numerous biotechnological applications such as production of biofuels and pharmaceuticals, and it can be also used for bioremediation of polluted water and wastewater. One of the highest limitations for its large-scale cultivation is that it cannot synthesize vitamins B1 and B12 which are expensive and they have to be added to media. This study revealed that E. gracilis can be grown for long time periods without the addition of vitamins B1 and B12 in the co-culture containing filamentous fungus Cladosporium westerdijkiae, and bacteria Lysinibacillus boronitolerans and Pseudobacillus badius. Growing of E. gracilis in such co-cultures without the addition of vitamins can dramatically reduce large scale cultivation costs. Moreover, C. westerdijkiae could be used in biotechnology for immobilization and effective harvesting of E. gracilis from big cultivation containers by bioflocculation.

Discrimination of Euglena gracilis strains Z and bacillaris by MALDI-TOF MS.

AIMS: Euglena gracilis is used as model organism for various microbiological, molecular biological and biotechnological studies. Its most studied wild-type strains are Z and bacillaris, but their discrimination by standard molecular methods is difficult. Therefore, we decided to test the suitability of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for identification of E. gracilis and for discrimination of these two strains possessing functional chloroplasts. MALDI-TOF MS profiling was also tested for two white (non-photosynthetic) stable E. gracilis mutant strains Wgm ZOflL and W10 BSmL. METHODS AND RESULTS: We have successfully obtained main spectrum profiles (MSPs) of E. gracilis strains Z, SAG 1224-5/25 and bacillaris, SAG 1224-5/15 using protein extraction procedure. Subsequent MALDI-TOF MS profiling of a number of tested samples and the comparison of the obtained protein profiles with our in-house database including MSPs of both strains have revealed that these two strains can be easily distinguished by MALDI-TOF MS based on score values over two in most cases. This method has also confirmed the ancestry of white mutant strains Wgm ZOflL and W10 BSmL, originally derived from strains Z and bacillaris, respectively. CONCLUSIONS: MALDI-TOF MS is suitable, accurate and rapid method for discrimination of E. gracilis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can have broad practical implications for laboratories cultivating various strains of euglenids, and they can be applied for their discrimination by MALDI-TOF MS.