DNA NUCLEOSIDE COMPOSITION AND METHYLATION IN SEVERAL SPECIES OF MICROALGAE1

Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed-phase high performance liquid chromatography. All samples contained 5-methyldeoxycytidine, although at widely varying levels. In M. minutum, about one-third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5′-CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae.     

TRANSPORT OF AMINO ACIDS BY THE SOIL ALGA STICHOCOCCUS BACILLARIS1

The green alga Stichococcus bacillaris Naeg. is able to take up at least eleven amino acids. All of these except glutamic and aspartic acids are transported by carrier systems that obey saturation kinetics. The acidic amino acids enter the cell by passive diffusion. Michaelis-Menten parameters (K_s and V_max) were calculated for several amino acids. All obey simple Michaelis-Menten behavior except for 2-methylalanine and leucine which may have double carrier systems of different affinities. Interactions between pairs of amino acids suggest that there is at least one carrier system specific for basic amino acids and probably several systems specific for neutral amino acids. Further analysis of neutral amino acid interactions reveal that the uptake of several amino acids is incompletely inhibited by competitor uptake at infinite concentration. The simplest interpretation of the data is the operation of three carrier systems for neutral amino acids, one of which has higher affinity and broader specificity than the other two. The amino acid carrier systems appear to operate by an active mechanism. The metabolic poison DCCD inhibits uptake up to 99%. The capacities of the neutral amino acid carrier systems are increased when cells are grown in medium containing suboptimal concentrations of nitrogen.     

Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure

Phylogenetic analyses, based upon nuclear small-subunit ribosomal RNA gene sequences, of four ‘chlorosarcinoid’ species referred to Planophila Gerneck show that the genus is polyphyletic. The type species, P. laetevirens Gerneck, is closely related to species in the Ulotrichales, Ulvophyceae. The monotypic sarcinoid genus Pseudendocloniopsis is the closest relative of Planophila; the two genera represent the addition of a new morphological type to the Ulotrichales. Planophila microcystis (Dangeard) Kornmann & Sahling forms a clade at the base of the Ulvophyceae with Oltmannsiellopsis, and thus belongs to the Oltmannsiellopsidales. This result is also supported by the Oltmannsiellopsis-like ultrastructure of P. microcystis zoospores. Planophila sp. B from Antarctica, which has Trebouxia-like pyrenoid structure, is a trebouxiophyte closely related to Chlorella-like unicellular coccoids, Stichococcus bacillaris and Prasiola species. This is the first robustly supported molecular phylogenetic analysis that places Prasiola in the Trebouxiophyceae. As shown previously, P. terrestris Groover & Hofstetter belongs to the Chaetopeltidales, Chlorophyceae. Dangemannia gen. nov. (type species : D. microcystis (Dangeard) comb. nov.), Floydiella gen. nov. (type species : F. terrestris (Groover & Hofstetter) comb. nov.) and Pabia gen. nov. (type: P. signiensis sp. nov.) are proposed.     

Inorganic carbon acquisition in two green marine Stichococcus species

The mechanism of inorganic carbon (C(i)) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of C(i) uptake indicate that both species have high apparent affinity for CO(2) with a low K(1/2) (CO(2)) of about 10 μm. The O(2) evolution rates in light exceeded the spontaneous CO(2) formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO(2) and O(2) fluxes showed that rates of O(2) evolution exceeded those of CO(2) depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO(2) upon illumination to a CO(2) compensation concentration of 15.42 and 12.03 μm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO(2) uptake: addition of bovine CA at CO(2) compensation concentration caused a rapid rise in CO(2) as the CO(2) -HCO(3) (-) equilibrium was restored. Accumulation of unfixed C(i) by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external C(i) concentration.     

A new aerial alga, Stichococcus ampulliformis sp. nov. (Trebouxiophyceae, Chlorophyta) from Japan

A new aerial alga, Stichococcus ampulliformis S. Handa sp. nov. (Trebouxiophyceae, Chlorophyta) is described based on a clone isolated from the bark of Cephalotaxus harringtonia (Knight ex Forbes) K. Koch collected from Taishaku‐kyo Gorge, Hiroshima Prefecture, south‐west Japan. This alga was examined by light microscopy and transmission electron microscopy and subjected to molecular phylogenetic analysis. Based on its morphological features and life‐cycle, especially short filament formation, the alga was assigned to the genus Stichococcus Nägeli. However, this alga differs from other described Stichococcus species in that it reproduces by a form of ‘budding’, producing two daughter cells of different sizes. The larger cell, enclosed within the mother cell wall, soon reaches the size of a normal vegetative cell. The smaller cell is extruded and takes longer to reach full vegetative size. A phylogenetic tree constructed using 18S rRNA sequences indicated that, within the Trebouxiophyceae, S. ampulliformis is closely related to S. bacillaris Nägeli and some species of Prasiola Meneghini.     

ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR‐ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM,HORMIDIELLA, AND ENTRANSIA1

Nuclear‐encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well‐supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as “Klebsormidium” emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384‐1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst’s morphology‐based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.     

NITROGEN METABOLISM AND AMINO ACID NUTRITION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

Nitrate-grown cells of Stichococcus bacillaris Naeg. (UTEX 314) contained much higher activities of glutamine synthetase (GS) and NADPH-glutamate dehydrogenase (GDH) than ammonium-grown cells. Methylamine, a non-metabolizable ammonium analog, caused a decrease in GS activity in nitrate-grown cells suggesting that GS is regulated by the size of the endogenous ammonium pool. The decrease in GS observed in methylammonium-loaded nitrate-grown cells was accompanied by an increase in NADPH-GDH activity. Stichococcus bacillaris can be grown in the presence of methionine sulfoximine (MSX), a potent inhibitor of GS. However, only a fraction of a control cell population showed a requirement for glutamine or arginine for growth following MSX addition. Fully adapted MSX-grown cells were indistinguishable from control cells in their ability to photosynthesize and utilize amino acids as nitrogen sources. Alanine, arginine, asparagine, glutamine, glycine and proline were good nitrogen sources, and maximum capacity for amino acid transport was developed in cells grown on these amino acids. Compared to nitrate-grown cells the activity of GS in ammo acid-grown cells was low, whereas NADPH-GDH was very active. The activity of NADH-GDH in amino acid-grown cells was highest under heterotrophic conditions.     

The marine algae of Rhodos island,Greece

The main purpose of this research is to describe the distribution of marine algae along the shores of Rhodos island. In the investigated medio- and infralittoral zones, collections revealed 131 species distributed as follows: Chlorophyceae 22·9%, Phaeophyceae 20·6% and Rhodophyceae 56·5%. The ratio Rhodophyceae/Phaeophyceae is 2·7, similar to that found by Gerloff & Geissler (1974) and lower than in other parts of the Mediterranean sea (Feldmann, 1938; Giaccone, 1968).     

Observations on tolerance to heavy metals of four green algae in relation to pH

Abstract

The inhibition of growth by different concentration of eight heavy metals: Cd, Cr, Co, Cu, Hg, Ni, Se and Tl, in inorganic medium at pH 3 and 6.5, was studied in four green algae: Chlorella protothecoides Krüger, Chlorella saccharophila (Krüger) Migula, Coenochloris sp. and Stichococcus bacillaris Naegeli.

The results suggest that pH has an important effect on heavy metal toxicity in algae although it is difficult to establish a relationship between pH and heavy metals.     

FERRIC CHELATE REDUCTASE ACTIVITY AS AFFECTED BY THE IRON‐LIMITED GROWTH RATE IN FOUR SPECIES OF UNICELLULAR GREEN ALGAE (CHLOROPHYTA)1

Four species of green algae (Chlorella kessleri Fott et Nováková, Chlorococcum macrostigmatum Starr, Haematococcus lacustris[Girod‐Chantrans] Rostaf., Stichococcus bacillaris Näg.) were grown in iron‐limited chemostats and under phosphate limitation and iron (nutrient) sufficiency. For all four species, steady‐state culture density declined with decreasing degree of iron limitation (increasing iron‐limited growth rate), whereas chl per cell or biovolume increased. Plasma membrane ferric chelate reductase activity was enhanced by iron limitation in all species and suppressed by phosphate limitation and iron sufficiency. These results confirm previous work that C. kessleri uses a reductive mechanism of iron acquisition and also suggest that the other three species use the same mechanism. Although imposition of iron limitation led to enhanced activities of ferric chelate reductase in all species, the relationship between ferric chelate reductase activity and degree of iron limitation varied. Ferric chelate reductase activity in C. macrostigmatum and S. bacillaris was an inverse function of the degree of iron limitation, with the most rapidly growing iron‐limited cells exhibiting the highest ferric chelate reductase activity. In contrast, ferric chelate reductase activity was only weakly affected by the degree of iron limitation in C. kessleri and H. lacustris. Calculation of ferric reductase activity per unit chl allowed a clear differentiation between iron‐limited and iron‐sufficient cells. The possible extension of the ferric chelate reductase assay to investigate the absence or presence of iron limitation in natural waters may be feasible, but it is unlikely that the assay could be used to estimate the degree of iron limitation.