We examined the morphology, physiology, and 16S rRNA gene sequences of three culture collection strains and of ten novel isolates of unicellular cyanobacteria from hypersaline environments. The strains were morphologically diverse, with average cell widths ranging from 2.8 to 10.3 micron. There were single-celled, colonial, and baeocyte-forming strains. However, morphological traits were markedly variable with culture conditions. In contrast, all strains displayed extreme halotolerance (growing close to optimally at above 12% salinity); all were obligately marine, euryhaline, and moderately thermophilic; and all shared a suite of chemotaxonomic markers including phycobilins, carotenoids, and mycosporine-like amino acids. 16S rRNA gene sequence analysis indicated that the strains were related to each other. Sequence similarity analysis placed the strains in a monophyletic cluster (which we named the Halothece cluster) apart from all cultured or uncultured, not extremely halotolerant cyanobacteria whose 16S rRNA gene sequences are available in public nucleotide sequence databases. This represents the first case in which a phylogenetically coherent group of cyanobacteria can be defined on the basis of physiology. The Halothece cluster contained two subclusters that may be divergent at the generic level, one encompassing 12 strains (spanning 5% 16S rRNA gene sequence divergence and named the Euhalothece subcluster), and a single deep-branching isolate. Phenotypic characterization of the isolates, including morphological, physiological, and chemotaxonomic traits, did not distinguish these subclusters and only weakly suggested the existence of two separate clades, one encompassing strains of small cell size (cell width < 5 m) and another one encompassing strains of larger cell size.

The phylogeny of unicellular, extremely halotolerant cyanobacteria.

The major organic osmoregulatory solutes of 36 cyanobacteria from a wide range of environmental sources have been examined using 13C nuclear magnetic resonance spectroscopy. These strains were also examined for their salt-tolerance, and could be arranged in three salt-tolerance groups, designated freshwater, marine and hypersaline. The most salt-tolerant cyanobacteria in the hypersaline group are properly classified as moderately halophilic. Cyanobacteria from all habitats and taxonomic groups accumulated organic osmoregulatory solutes, and the chemical class of the solute correlated with the salt-tolerance and habitat of the strain. Freshwater strains accumulated simple saccharides, predominantly sucrose and trehalose; marine strains accumulated the heteroside O-α-D-glucopyranosyl-(1→2)-glycerol, and hypersaline strains accumulated sucrose and/or trehalose together with glycine betaine or the novel solute L-glutamate betaine (N-trimethyl-L-glutamate) or they accumulated glycine betaine alone. The results suggest that the presence of certain major organic osmoregulatory solutes may be useful in the numerical taxonomy of cyanobacteria, and in the identification of some ionic characteristics of the environment of origin of each isolate.

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Organic Osmoregulatory Solutes in Cyanobacteria

The oxygenic photosynthetic procaryotes comprise a single taxonomic and phylogenetic group (see master phylogenetic tree of the Bacteria). In the last edition of the Manual, two separate groups were described, but it is now apparent that members of the Prochlorales simply represent different, unrelated genera which fall into the main cluster of the Cyanobacteria (see Oxygenic Photosynthetic Bacteria, below). The principal character that defines all of these oxygenic photosynthetic procaryotes is the presence of two photosystems (PSII and PSI) and the use of H2O as the photoreductant in photosynthesis. Although facultative photo- or chemo-heterotrophy may occur in some species or strains, all known members are capable of photoautotrophy (using CO2 as the primary source of cell carbon).

Phylum BX. Cyanobacteria

Publisher Summary Microalgae are of very great ecological importance as the major primary producers in large bodies of water. They are also found in benthic habitats, in soil, and in a number of symbioses, particularly corals and lichens. It discusses that microalgae may not seem to be the organisms of choice for investigating the mechanism of the transport of nutrients across the plasmalemma—they are usually photolithotrophs, which poses problems both for the growth of the organisms and for unravelling the energy sources for transport. In addition to the respiratory and fermentative energy sources of heterotrophic cells, there is also the possibility of direct intervention of photoproduced high-energy compounds in powering transport in the light. It has not so far proved easy to separate the plasmalemma from other cell membranes in subcellular preparations, even in the structurally simple cyanobacteria. This is a prerequisite for detailed analysis of membranes and characterization of the transport systems found in them. Their ecological importance and the possibility of testing current chemiosmotic views on the mechanism of membrane transport in organisms, which live at extremes of pH and of osmotic pressure make them very important experimental objects.

Nutrient Transport in Microalgae

Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology

The intracellular ion content of the halophilic blue-green alga, Aphanothece halophytica was studied as a function of age, external sodium and external potassium concentration. Intracellular Na+ was found to be about 0.38 millimoles/g dry mass. Intracellular K+ concentrations were as high as 1 M and varied directly with external salinity. Intracellular Ca++ and Mg++ were in the range previously reported for fresh water blue-green algae despite their extremely high extracellular concentrations. Average cell size is consistent at room temperature with two exceptions. When the outside K+ is lower than 6.5 mM the cells tend to be smaller with less intracellular K+ and high Ca++. In stationary phase cultures the cells are larger with high intracellular Mg++ and low K+.

Ion metabolism in a halophilic blue-green alga, Aphanothece halophytica.

Seventeen clones of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi and Fukuyo were acclimated to the same environments over several months. Significant variance components were detected between non-acclimated and acclimated cultures for cell potencies, yields and reproduction rates. The resultant variance in acclimated potencies among clones was statistically significant (P < 0.0001), indicating that potency can be used for genetic comparisons. However, cell potency differences for a clone of G. toxicus in the acclimated vs. non-acclimated phases can exceed genetic differences between clones. This stresses the need for a rigorous acclimation process. Caribbean isolates of G. toxicus were inherently more toxic than isolates from other areas. One Caribbean clone yielded 55 × 10−4 mu (mouse units)·cell−1 whereas clones Bermuda, the Bahamas, and Florida ranged from only 1.8 × 10−4 mu·cell−1 to a maximum of 19.8 × 10−4 mu·cell−1. Toxicity decreased with increasing latitude (r =–0.819, P < 0.01), indicating that environmental differences probably influenced the potencies. A comparison of acclimated reproduction rates at four light intensities also indicated that genetic differences among clones existed. The resulting reproduction rate/light slopes overlapped, indicating that the clones may be adapted to specific light regimes.

Genitic variability in toxin potencies among seventeen clones of gambierdiscus toxicus (dinophyceae)1

An axenic culture of an extremely halotolerant blue-green alga, Aphanothece halophytica (Chroococcaies), was obtained following a multi-step purification procedure. Effectiveness of purification procedures as a function of hypersalinity is discussed. Development of a defined medium and growth in NaCl up to saturation is reported. Changes in cellular morphology are concomitant to changes in salinity and other physico-chemical parameters. Lysis of the alga in distilled water and in media of less than 1 m NaCl, as well as failure of KCL, LiCl and CsCl to substitute for NaCl support the view that the alga is perhaps halophilic rather than only halotolerant.

Isolation, purification and evidence for a halophilic nature of the blue-green alga Aphanothece halophytica Fremy (Chroococcales)

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.

Protein and amino acid composition of the obligate halophile aphanothece halophytica (cyanophyta)1

An atlas of the cerebral ganglion of Moniliformis dubius has been constructed and the cells identified by number. There is a total of 88 cells, two of which are binucleate. These cells (20 and 90) are located in the frontal plane. The cell population is equally distributed between each half of the ganglion, there being 41 cells on each side with six cells centrally located between the two halves. Most cells give rise to bilateral processes and are organized in such a way that there is an outer coat of somata or cell bodies. Cell bodies predominate on the ventral surface, but the dorsal surface is more of a mixture of cell bodies and nerve processes. The core or neuropile occupies the central part of the ganglion and is the site for cross over for many cell processes which then exist from the side opposite the cell body. The cytoarchitecture of these cells varies considerably. Most have a large round nucleus with well defined nucleoli.

Donald M. Miller

Papers

Ion metabolism in a halophilic blue-green alga, Aphanothece halophytica.

Genitic variability in toxin potencies among seventeen clones of gambierdiscus toxicus (dinophyceae)1

Isolation, purification and evidence for a halophilic nature of the blue-green alga Aphanothece halophytica Fremy (Chroococcales)

Protein and amino acid composition of the obligate halophile aphanothece halophytica (cyanophyta)1

Anatomy of the cerebral ganglion of the male acanthocephalan, Moniliformis dubius.