After the description of Chlorella vulgaris by Beijerinck, 120 years ago, members of the genus Chlorella belong to the best studied green algae worldwide. However, numerous open questions remained regarding their systematics. Recent molecular studies showed the polyphyly of the genus within the Chlorophyceae and Trebouxiophyceae. Chlorella-species were traditionally characterized by spherical to oval cell shape, solitary lifeform and the absence of mucilaginous envelopes. The challenge in the past was how to distinguish species due to their high phylogenetic diversity combined with a limited amount of morphological characters. Using a polyphasic approach of SSU- and ITS rDNA phylogeny, secondary structure of the ITS and light microscopic observations, we were able to detect six lineages with Dictyosphaerium-like strains in close relationship to C. vulgaris, here described or combined newly as C. coloniales sp. nov., C. pituita sp. nov., C. pulchelloides sp. nov., C. singularis sp. nov., C. elongata comb. nov. and C. chlorelloides comb. nov. Furthermore, three new species without mucilage were described as C. lewinii sp. nov., C. rotunda sp. nov. and C. volutis sp. nov. Using the 5.8S rRNA and part of the ITS-2 as molecular signature (barcode), we were able to distinguish not only the five already known species of Chlorella, C. vulgaris, C. sorokiniana, C. heliozoae, C. lobophora and C. variabilis but the seven new species and two new combinations as well. CBCs and hemi-CBCs within the secondary structure of the ITS-2 confirmed the separation of the species. Our study led to a new understanding of the evolution of morphology within the genus Chlorella and to an emendation of the generic description.

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Taxonomic reassessment of the genus Chlorella (Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species.

Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple "extracellular" layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes.

https://www.pnas.org/content/pnas/103/1/165.full.pdf

The Bacillus subtilis spore coat provides “eat resistance” during phagocytic predation by the protozoan Tetrahymena thermophila

Marine natural product peptides with therapeutic potential: Chemistry, biosynthesis, and pharmacology

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.

Endosymbionts in Paramecium

Papain-like cysteine peptidases are a diverse family of peptidases found in most known organisms In eukaryotes, they are divided into multiple evolutionary groups, which can be clearly distinguished on the basis of the structural characteristics of the proenzymes Most of them are endopeptidases; some, however, evolved into exopeptidases by obtaining additional structural elements that restrict the binding of substrate into the active site In humans, papain-like peptidases, also called cysteine cathepsins, act both as non-specific hydrolases and as specific processing enzymes They are involved in numerous physiological processes, such as antigen presentation, extracellular matrix remodeling, and hormone processing Their activity is tightly regulated and dysregulation of one or more cysteine cathepsins can result in severe pathological conditions, such as cardiovascular diseases and cancer Other organisms can utilize papain-like peptidases for different purposes and they are often part of host-pathogen interactions Numerous parasites, such as Plasmodium and flukes, utilize papain-like peptidases for host invasion, whereas plants, in contrast, use these enzymes for host defense This review presents a state-of-the-art description of the structure and phylogeny of papain-like peptidases as well as an overview of their physiological and pathological functions in humans and in other organisms

Papain-like peptidases: structure, function, and evolution

The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 107 algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 103 algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.

Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria

The green paramecium, Paramecium bursaria, has endosymbiotic algae in the cytoplasm. Here, we report that all endosymbiotic algae are destroyed, producing symbiotic algae-free strains of paramecia, when green paramecia are cultivated in the presence of a herbicide paraquat.

https://bioone.org/journals/zoological-science/volume-12/issue-6/zsj.12.807/Symbiotic-Algae-free-Strains-of-The-Green-Paramecium-Paramecium-bursaria/10.2108/zsj.12.807.pdf

Symbiotic Algae-free Strains of The Green Paramecium Paramecium bursaria Produced by Herbicide Paraquat

A protease in the culture medium of Tetrahymena pyriformis was purified to homogeneity. The purified protease had an apparent molecular mass of 28 kDa on SDS/PAGE. The amino acid sequences of the N-terminal and internal peptides of the protease showed complete identity with those of tetrain, an enzyme previously reported as a Tetrahymena cysteine protease but not characterized in detail. Two overlapping cDNA clones for tetrain were sequenced, and the nucleotide sequence predicts that these clones encode a 330-amino acid protein composed of a 16-residue N-terminal signal sequence followed by a 103-residue propeptide and a 211-residue mature protease. The primary structure and enzymatic properties support the conclusion that tetrain belongs to the cathepsin L subfamily. Immunoblotting analyses showed that mature tetrain was found exclusively in the culture medium. Immunofluorescence microscopy demonstrated that tetrain was concentrated in or around the food vacuoles of cells in the late logarithmic phase, but the staining of food vacuoles was not obvious in the stationary phase. These results suggest that tetrain is synthesized at the logarithmic phase and is secreted into the culture medium as a mature form.

Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily

Characterization of Symbiotic Algae-free Strains of Paramecium bursaria Produced by the Herbicide Paraquat

Paramecia demonstrate rotational cytoplasmic streaming, in which some cytoplasmic granules and organelles, including symbiotic algae, flow in a constant direction. To elucidate the mechanism of this streaming, we examined the effects of cytochalasins (cytochalasin B and D, and dihydrocytochalasin B) and nocodazole, which are reagents affecting microfilament and microtubule networks, respectively, in the cell. In previous reports, paramecia have been compressed with a coverslip to facilitate observation of cytoplasmic streaming. Here we found that the cytoplasmic streaming of paramecia was suppressed by such compression and then observed the process without compression in this work. In the presence of cytochalasins, cytoplasmic streaming was not affected. In contrast, treatment with nocodazole (10 μg/ml) resulted in discontinuation of cytoplasmic streaming in paramecia. Immunofluorescent microscopic observations by confocal microscopy revealed that the number of intracellular microtubules in nocodazole-treated cells was markedly decreased compared to that of controls. Electron microscopic observations confirmed the decrease. These results suggest that cytoplasmic microtubules play an important role in the cytoplasmic streaming of paramecia. Cell Motil. Cytoskeleton 43:85–98, 1999. © 1999 Wiley-Liss, Inc.

Tadao Takahashi

Papers

Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria

Symbiotic Algae-free Strains of The Green Paramecium Paramecium bursaria Produced by Herbicide Paraquat

Secretion of tetrain, a Tetrahymena cysteine protease, as a mature enzyme and its identification as a member of the cathepsin L subfamily

Characterization of Symbiotic Algae-free Strains of Paramecium bursaria Produced by the Herbicide Paraquat

Microtubule-dependent movement of symbiotic algae and granules in Paramecium bursaria.