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HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P2(1)2(1)2(1) grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 A resolution. HML is the first marine alga lectin to be crystallized.
The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified into three genera, Cyanidium, Cyanidioschyzon and Galdieria. Little is known, however, about the biodiversity of Cyanidiales, their population structure and their phylogenetic relationships. Here we used a taxonomically broadly sampled three-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp. and the Cyanidioschyzon merolae plus Galdieria maxima lineage. Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. We also used environmental polymerase chain reaction (PCR) for the rbcL gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution.
Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42,000-Mr subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of Mr 470,000 and apparent subunit composition X8Y4. The subunits X and Y are of Mr 39,000 and 42,000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD. Depolymerisation of the latent phosphoribulokinase to give the low-Mr form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol. The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-Mr latent form of the enzyme.
The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.
The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.
Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb. purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the L1637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP+ reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.
The pathogenicity of strains of yeasts and algae isolated from bovine mastitis secretions was evaluated in immunosuppressed mice. Strains of Candida tropicalis (n = 3) were the most pathogenic, but all strains of Geotrichum capitatum (n = 5) and the colourless alga Prototheca zopfii (n = 5) were also lethal to mice at the highest dose of 1 x 10(7) CFU per mouse. Reisolation of the inoculated micro-organisms and the occurrence of histopathological lesions within organs of mice challenged with 1 x 10(3) to 1 x 10(7) CFU per animal revealed strains of C. krusei (n = 17), C. kefyr (n = 6) and C. rugosa (n = 6) to have a moderate pathogenicity, whereas strains of C. valida (n = 1) and C. catenulata (n = 2) were weakly pathogenic and non-pathogenic respectively.
The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.
The two ycf27 genes from the filamentous cyanobacterium Tolypothrix PCC 7601 have been cloned and sequenced. These two genes, previously designated rpaA and rpaB, encode putative transcriptional regulators of the 'OmpR' family. In Synechocystis PCC 6803, homologous genes have been linked to the regulation of transfer of excitation energy from the phycobilisome to photosystem (PS) I and PSII respectively. Partial clones from Spirulina platensis, Dactylococcopsis salina and Synechococcus PCC 7002 have also been sequenced. A table of identity between the proteins confirms that RpaB belongs in the same family as the algal ycf27 proteins. However, RpaA is a rather different protein and should lose the designation ycf27. The loss of rpaB from the plastid genomes of eukaryotic algae is associated with the loss of phycobiliproteins, so it is likely that this gene performs a similar role in algae to that in cyanobacteria. The implications for chloroplast evolution are discussed along with the possible identity of the cognate histidine kinase gene in the plastid genomes.
Silicified peritidal carbonate rocks of the 1250- to 750-million-year-old Hunting Formation, Somerset Island, arctic Canada, contain fossils of well-preserved bangiophyte red algae. Morphological details, especially the presence of multiseriate filaments composed of radially arranged wedge-shaped cells derived by longitudinal divisions from disc-shaped cells in uniseriate filaments, indicate that the fossils are related to extant species in the genus Bangia. Such taxonomic resolution distinguishes these fossils from other pre-Ediacaran eukaryotes and contributes to growing evidence that multicellular algae diversified well before the Ediacaran radiation of large animals.
Ribosomal RNA from several blue-green algae was hybridized to DNA from Euglena gracilis chloroplasts by the membrane filter procedure. This hydridization was competitive with chloroplast ribosomal RNA and indicates significant genetic homology between blue-green algae and plastids from Euglena gracilis.
The injection of carbon dioxide and the addition of nitrogen and phosphorus to a lake population dominated by blue-green algae results in a rapid shift to dominance by green algae. The basis for the change and its implications are discussed.
Predawn episodes of mass spawning by green algae (up to nine species in five genera on a single morning) intermittently cloud Caribbean waters. Species- and sex-specific bouts of anisogamous gamete release occurred synchronously and predictably on a given morning, with closely related species spawning at different times. Algal sexual reproduction was seasonal, but, unlike the mass-spawning behavior of other sessile marine organisms, showed no lunar or tidal cycling. The discovery of mass-spawning behavior by these algae has important implications for future studies of the reproductive ecology and speciation of a vital, yet poorly understood, component of the coral reef community.
Ten species of freshwater blue-green algae exhibit an adenosylcobalamin-dependent ribonucleotide reductase, thuse explaining the requirement for cobalt by these organisms. The evidence suggests a phylogenetic affinity between the cyanophytes and bacteria, such as Clostridium and Rhizobium, and the euglenoid flagellates, which also use the cofactor-dependent reductase. In contrast, the ribonucleotide reductase reaction in the few green algae surveyed shows no dependence on cobalamins.
Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.
The nutritional characteristics of an alga (Microcystis sp.) that occurs naturally in a Guatemalan lake were determined. The sun-dried material proved to have a high protein content (55.6%) and to be a possible good source of calcium and phosphorus (1, 169.1 and 633.4 mg/100 mg, respectively). Amino acid analysis showed that total sulfur amino acids were the most deficient ones, giving a protein score of 42 to the material. The in vitro protein digestibility of the material was 69.5%. Biological trials demonstrated that when the material was offered as the only protein source, very low consumption and a high mortality rate were obtained whether or not the diet was supplemented with 0.4% dl-methionine. However, when the material supplied 25% of the total protein of a corn-algae diet, the protein quality of the cereal was significantly improved (P < 0.05).
An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5'-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described.
Determinations of the number of microorganisms in lake water samples with the bright-field light microscope were performed using conventional counting chambers. Determinations with the fluorescence microscope were carried out after staining the organisms with acridine orange and filtering them onto Nuclepore filters. For transmission electron microscopy, a water sample was concentrated by centrifugation. The pellet was solidifed in agar, fixed, dehydrated, embedded in Epon, and cut into thin sections. The number and area of organism profiles per unit area of the sections were determined. The number of organisms per unit volume of the pellet was then calculated using stereological formulae. The corresponding number in the lake water was obtained from the ratio of volume of solidified pellet/volume of water sample. Control experiments with pure cultures of bacteria and algae showed good agreement between light and electron microscopic counts. This was also true for most lake water samples, but the electron microscopic preparations from some samples contained small vibrio-like bodies and ill-defined structures that made a precise comparison more difficult. Bacteria and small blue-green and green algae could not always be differentiated with the light microscope, but this was easily done by electron microscopy. Our results show that transmission electron microscopy can be used for checking light microscopic counts of microorganisms in lake water.