Fine Structure of the Cell Surface and Golgi Apparatus of Ochromonas*

SYNOPSIS. Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340–360 nm long, and small projections, 50–110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthenium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

Element Stoichiometry of a Mixotrophic Protist Grown Under Varying Resource Conditions

ABSTRACT. The balance of essential elements (e.g. carbon [C], nitrogen [N], and phosphorus [P]) between consumers and their resources influences not only the growth and reproduction of the consumers but also the nutrients they regenerate. Flagellate protists are significant predators of aquatic bacteria and directly influence nutrient flow to higher trophic levels and, through excretion, influence the mineral element composition of dissolved nutrients. Because the element stoichiometry of protists is poorly characterized, we varied the resource composition of the bacterium Pseudomonas fluorescens and used it to grow the mixotrophic bacterivorous flagellate Ochromonas danica. Using a mass balance approach, the element composition of O. danica was found to vary depending upon the nutrient composition of the prey and ranged between 482:36:1 and 80:12:1 (C:N:P molar). Homeostasis plots suggested that flagellate protists weakly regulate their element composition and are likely to regenerate different elements depending upon the nature of the element limiting growth of their prey.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Research note: Analysis of expressed sequence tags from the chrysophycean alga Ochromonas danica (Heterokontophyta)

A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′‐end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E‐value <10^?4) to sequences in a public database. The annotation of these ESTs was carried out to assess subcellular localization of the putative proteins using several internet‐accessible prediction programs for subcellular localization. These analyses revealed that putative plastid proteins in Ochromonas possess N‐terminal bipartite presequences with a conserved phenylalanine at the N‐terminus of the predicted transit peptide‐like domains, similar to other ‘red‐lineage’ secondary symbiotic organisms. The examination of sequences of 3′‐UTR revealed that, similarly to chlorophyte algae, UGUAA may represent a putative polyadenylation signal in O. danica.     

Complete large subunit ribosomal RNA sequences from the heterokont algae ochromonas danica, nannochloropsis salina, and tribonema aequale, and phylogenetic analysis

The large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale were determined. These sequences were combined with small subunit ribosomal RNA sequences in order to carry out a phylogenetic analysis based on neighbor-joining, maximum parsimony, and maximum likelihood methods. Our results indicate that heterokont fungi and heterokont algae each are monophyletic, and confirm that they together form a monophyletic group called ``stramenopiles.' Within the heterokont algae, the eustigmatophyte Nannochloropsis salina either clusters with the chrysophyte Ochromonas danica or forms a sister group to a cluster comprising the phaeophyte Scytosiphon lomentaria and the xanthophyte Tribonema aequale. The alveolates were identified as the closest relatives of the stramenopiles, but the exact order of divergence between the eukaryotic crown taxa could not be established with confidence. Received: 22 November 1996 / Accepted: 14 February 1997     

Characterization of centrin genes from Ochromonas danica (Chrysophyceae) and Scytosiphon lomentaria (Phaeophyceae)

Centrin, the EF‐hand Ca²⁺‐binding protein is localized at the basal apparatus of flagella and in centrioles in many eukaryotic cells. In the present study, centrin genes of the heterokont algae have been clarified for the first time. We isolated and analyzed cDNA and genomic DNA of centrin genes from the crysophycean alga Ochromonas danica Prings (UTEX LB1298) and the brown alga Scytosiphon lomentaria (Lyngbye) Link. The centrin gene of Ochromonas contained an open reading frame of 163 amino acids. The deduced protein, named Odcen, exhibited 85%, 78% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. The centrin genes of Scytosiphon contained an open reading frame of 164 amino acids. The deduced protein, named Slcen, exhibited 84%, 77% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. Both Odcen and Slcen possessed N‐terminal extensions before the conserved amino acid among various centrins, four EF‐hand domains and an aromatic amino acid at the C‐terminus. Southern blot hybridization suggested that the centrin gene occurs as a single copy gene in both Ochromonas and Scytosiphon genomes. Comparison of the sequence of the cDNA and the genomic DNA revealed that the Odcen gene was split into three fragments by introns and Slcen gene consisted of five fragments. The junctions of all introns of both genes conformed to the GT–AG rule. The introns of Slcen gene were considerably long and, as a result, the Slcen gene was approximately seven times longer than Odcen gene.     

A TUBULAR MASTIGONEME‐RELATED PROTEIN,OCM1, ISOLATED FROM THE FLAGELLUM OF A CHROMOPHYTE ALGA,OCHROMONAS DANICA1

The phylogenetic group stramenopiles refers to the systematic groups that possess tripartite tubular hairs (stramenopiles) on their flagella. There have been a number of studies describing the fine structure of these mastigonemes and a few studies isolating the component proteins; however, these proteins and their gene sequences have not yet been identified. In the present study, we identified a mastigoneme protein (Ocm1) of the chrysophycean alga Ochromonas danica Pringsh. (UTEX LB1298). Its corresponding gene, Ocm1, was identified by using degenerate primers that correspond to the partial amino acid sequences of a protein (85 kDa) obtained from a mastigoneme‐rich fraction of isolated flagella. The polypeptide encoded by Ocm1 has four cysteine‐rich, epithelial growth factor (EGF)–like motifs, potentially involved in protein–protein interactions. It lacks obvious hydrophobic regions characteristic of transmembrane domains, suggesting that this polypeptide is not likely a protein for anchoring the mastigoneme. In addition, a polyclonal antibody against Ocm1 labeled the area where the tubular shafts of the mastigonemes are located, but not the basal portion or the terminal filaments.     

Phagotrophic Ingestion of a Blue-Green Alga by Ochromonas*†

Anacystis nidulans disappeared rapidly from culture in the presence of an unidentified species of Ochromonas. Disappearance was light-independent and could be induced neither by bacteria associated with, nor by soluble products released from the flagellate. Electronmicrographs of mixed cultures revealed numerous A. nidulans cells in various stages of digestion within vacuoles of Ochromonas. Evidently the disappearance of the alga from culture resulted from phagotrophy by the chrysomonad. A 2-stage digestive process is suggested whereby A. nidulans cells are initially sequestered in the posterior “leucosin” vacuole and then undergo the terminal stages of digestion and elimination in smaller, peripheral vacuoles.     

The costs and benefits of multicellular group formation in algae*

The first step in the evolution of complex multicellular organisms involves single cells forming a cooperative group. Consequently, to understand multicellularity, we need to understand the costs and benefits associated with multicellular group formation. We found that in the facultatively multicellular algae Chlorella sorokiniana: (1) the presence of the flagellate Ochromonas danica or the crustacean Daphnia magna leads to the formation of multicellular groups; (2) the formation of multicellular groups reduces predation by O. danica, but not by the larger predator D. magna; (3) under conditions of relatively low light intensity, where competition for light is greater, multicellular groups grow slower than single cells; (4) in the absence of live predators, the proportion of cells in multicellular groups decreases at a rate that does not vary with light intensity. These results can explain why, in cases such as this algae species, multicellular group formation is facultative, in response to the presence of predators.     

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)1

Two‐dimensional (2‐D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine‐rich, epithelial growth factor (EGF)‐like motifs, potentially involved in protein–protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up‐regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.     

Particle aggregation and phagotrophy by Ochromonas

Summary Ochromonas danica creates a current with its flagella which forces fluid and particles to flow toward the front and over the sides of the phytoflagellate. It is able to aggregate particles including bacteria by this action, trap them at its surface, and engulf them. Aggregation and phagocytic activity requires live, metabolically active cells. This type of biological activity may permit O. danica and similar organisms to sample their environment and extract particles.Aided by grant GB 20825 from the National Science Foundation and 5-S05-RR-07064-06 from the National Institutes of Health.     

Effect of Aging of a Cell Population on Lipids and Drug Resistance in Ochromonas danica*

SYNOPSIS. Stationary cultures of Ochromonas danica accumulated lipids as they aged. The bulk of the increase in fatty acids was in the unsaturates, particularly the polyunsaturates. The quantity of lipid peroxides (thiobarbituric acid-positive material) also increased with age. Aging was also associated with increase in sensitivity to inhibitory compounds. The implications of lipid accumulation for cell sensitivity are discussed.     

Excretion of S- and O-methyl esters and other volatile compounds by Ochromonas danica

The formation of volatile excretion products was studied in axenic cultures of Ochromonas danica. Under microaerobic conditions in the light, an ac     

Comparative analysis of diacylglyceryl-trimethylhomoserine in Ochromonas danica and in Chlamydomonas reinhardtii 137+

The fatty acid compositions of the ether lipid 1(3),2-diacylglyceryl-(3)-O-4′-(N,N,N-trimethyl)homoserine (DGTS) from Ochromonas danica a     

Regulation of the distribution of excitation energy in Ochromonas danica,an organism containing a chlorophyll-A/C/carotenoid light harvesting antenna

A chlorophyll a, c-fucoxanthin pigment-protein complex8 functions as the major light harvesting antenna in the Chrysophyte Ochromonas danica. The regulated distribution of excitation energy between the two photosystems was investigated in these organisms and was shown to be strongly wavelength dependent. A light state transition was induced by pre-illumination of cells using light 2 (640 nm) and light 1 (700 nm) of equal absorbed intensity, and detected by reversible changes in the 77 K chlorophyll fluorescence emission spectra. Peaks at 690 nm and 720 nm in the low temperature spectra are most likely associated with PS2 and PS1 respectively. A room temperature fluorescence emission at 680 nm induced by modulated light 2 (500 nm) was strongly quenched in the presence of background light 1 (720 nm). Removal of light 1 led to an increase in fluorescence followed by a slow quenching. The room temperature fluorescence changes were directly correlated with changes in the 77 K emission spectra that indicated a change in the distribution of excitation energy between the two photosystems. It was established that DCMU (1 mol) prevented the state 2. The conversion to state 1 followed a simple photochemical dose dependence and had a half-time of 20 s-1.5 min at 6 W m^-2. In contrast, the conversion to state 2 was independent of light intensity. These data indicate that O. danica undergoes a light state transition in response to the preferential excitation of PS2 or PS1.Abbreviations PS2 photosystem 2 - PS1 photosystem 1 - LHC light harvesting chlorophyll a/b protein - fx fucoxanthin - PQ plastoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea     

Occurrence of a 3′-phosphoadenosine 5′-Phosphosulphate synthesizing system In two Ochromonas species

The presence of an enzyme capable of incorporating ³⁵SO₄^2? into 3′-phosphoadenosine 5′-phosphosulphate has been demonstrated,in Ochromonas danica and O. malhamensis. This system probably includes the enzymes ATP:sulphate adenyltransferase. E.C. 2.7.7.4 and ATP:adenylsulphate 3′-phosphotransferase, E.C. 2.7.1.25.     

Disproving the hypothesis of a common ancestry for the Ochromonas danica chrysoplast and Heliobacterium chlorum

The phylogenetic position of the golden-yellow alga Ochromonas danica chrysoplast was investigated by comparison of the 16S rRNA catalogue and two long 16S rRNA stretches (804 and 454 bases) with catalogues from eubacteria and chloroplasts and with homologoes 16S rRNA regions from Escherichia coli, Bacillus subtilis, Heliobacterium chlorum, Anacystis nidulans and chloroplasts from Zea mays, Nicotiana tabacum, Euglena gracilis and Chlamydomonas reinhardii, respectively. Both approaches indicate a closer relationship of the chrysoplast to chloroplasts and cyanobacteria than to the brownish photoheterotrophic Heliobacterium chlorum for which a common ancestry has recently been hypothesized.     

Ethionine and Methionine Metabolism by the Chrysomonad Flagellate Ochromonas danica

SYNOPSIS. Growth of Ochromonas danica is competitively inhibited by ethionine. Inhibition can be reversed by methionine. Inhibition indexes of the effect of ethionine on growth and methionine incorporation into proteins are 1 and 4, respectively. Inside the cell, methionine is partially de-methylated and metabolized to form cysteine. Ethionine is partially de-ethylated, and the homocysteine moiety is either re-methylated to form methionine or further metabolized to form cysteine. Ethionine is also incorporated into proteins of O. danica. The kind of metabolic interference, expressed by inhibition of growth, and correlated with incorporation of ethionine, is yet unknown.     

Dynamics and Nutritional Ecology of a Nanoflagellate Preying Upon Bacteria

Ingestion and growth rates of the nanoflagellate predator Ochromonas danica feeding on the bacterium Pseudomonas fluorescens were quantified in laboratory cultures. Bacterial prey were grown under four nutritional conditions with respect to macronutrient elements: C-limited, N-limited, P-limited, and balanced. Ingestion and growth rates were saturating functions of prey abundance when preying upon nutritionally balanced, C-limited, and P-limited bacteria but were unimodal functions of abundance when preying on N-limited bacteria. At saturating prey concentrations, the ingestion rate of C-limited prey was about twice that of prey in other nutritional states, while at subsaturating prey concentrations, the ingestion rates of both C- and N-limited prey were higher than those of prey in other nutritional states. Over all prey concentrations, growth was most rapid on balanced and C-limited prey and generally lowest for P-limited prey. Due to the unimodal response of growth rate to abundance of N-limited prey, growth rate on N-limited prey approached that obtained on balanced and C-limited prey when prey were available at intermediate abundances. The accumulation of recycled N increased with the growth rate of O. danica. Recycling of N was highest when O. danica was feeding upon P-limited prey. The accumulation of recycled P increased with growth rate for balanced and N-limited prey, but not for P-limited prey, which consistently had low accumulation of recycled P. The low growth rate and negligible recycling of P for O. danica preying on P-limited prey is consistent with the theory of ecological stoichiometry and resembles results found for crustacean zooplankton, especially in the genus Daphnia. Potentially, the major predators of bacterioplankton and a major predator of phytoplankton play analogous roles in the trophic dynamics and biogeochemistry of aquatic ecosystems.