Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

CLONING OF A cDNA ENCODING A 66-kDa Ca2+-DEPENDENT PROTEIN KINASE (CDPK) FROM DUNALIELLA TERTIOLECTA (CHLOROPHYTA)

A cDNA clone encoding a Ca²+-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1 , a CDPK from the green alga, Chlamydomonas eugamentos Moewus . The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca²+-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca²+ concentration in vivo .     

Solubilization and spectral characteristics of chlorophyll-protein complexes isolated from the thermophilic blue-green alga Synechococcus lividus

The thermophilic blue-green alga Synechococcus lividus was grown at 38 and 55°C. The reaction center chlorophyll-protein complexes (CP) of Photosystem (PS I) and PS II, CP a_I and CP a_II, were isolated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis at 4°C. SDS solubilization of thylakoids was performed in the temperature range 0–65°C. The low-temperature absorption and fluorescence emission spectral properties of the isolated chlorophyll-protein complexes were analyzed. Only traces of CP a_I were solubilized at temperatures below the lipid phase transition temperature. Instead, a minor PS I component, CP a′_I, was obtained that had absorption and fluorescence characteristics similar to those of CP a_I. CP a′_I had a slightly lower mobility than CP a_I in SDS-polyacrylamide gel electrophoresis. The amount of CP a_I in the gel scan profile increased dramatically when solubilization was carried out above the phase transition temperatures, but started to decrease above 60°C. CP a_II, on the other hand, could be efficiently extracted even at 0°C and was stable in the scan profile up to extraction temperatures of 30–40°C. Low-temperature absorption and fluorescence emission spectra were typical for CP a_I and CP a_II and no specific effects of the two growth temperatures on these properties were observed. The phase transition temperature was considered to be critical for the solubilization of CP a_I, either because of the difficulties of SDS (especially as it forms micelles at low temperatures) in penetrating the solidified membrane lipids at temperatures below that of the phase transition or because the CP a_I monomers of the PS I antennae are so strongly bound to each other that they cannot be dissociated by SDS before thermal agitation has reached a certain level that is achieved above the phase transition temperature. We consider both the difficulties in solubilizing CP a_I at sub-transition temperatures and the heat stability of the two complexes as adaptations which enable Synechococcus to grow under extreme high-temperature regimes.     

A selenium-induced peroxidation of glutathione in algae

Two unicellular marine algae cultured in media containing sodium selenite were examined for glutathione peroxidase activity. The 400 g supernatant from disrupted cells of both the green alga Dunaliella primolecta and the red alga Porphyridium cruentum were able to enhance both the H₂O₂ and the tert-butyl hydroperoxide dependent oxidation of glutathione. The glutathione peroxidation activity of D. primolecta was reduced only slightly by heating the 400 g supernatant, a 30% decrease in the rate with H₂O₂ and 10% decrease in the rate with t-BuOOH being observed. Heating caused the H₂O₂ dependent activity in P. cruentum to be reduced by only 30%, but the activity with t-BuOOH was reduced by 90%. Freezing decreased the t-BuOOH dependent activity of P. cruentum by 90%, but did not lower the t-BuOOH dependent activity of D. primolecta or the H₂O₂ dependent activity of either alga. It was concluded that the heat and cold stable, glutathione peroxidation was non-enzymatic in nature. A variety of small molecules (ascorbate, Cu(NO₃)₂, selenocystine, dimethyldiselenide and selenomethionine) were shown to be able to enhance the hydroperoxide dependent oxidation of glutathione in the assay system employed in this study. Such compounds could be responsible for the activity observed in algae. The heat and cold labile t-BuOOH reductase activity of P. cruentumwas possibly enzymatic, but was not attributable to the presence of glutathione-S-transferase. Both algae, when cultured in the presence of added selenite, displayed an approximate doubling of the non-enzymatic H₂O₂ and t-BuOOH dependent glutathione oxidase activities. The heat and cold labile t-BuOOH reductase activity of P. cruentum was unaltered when the alga was grown in the presence of added selenite. These observations are consistent with the hypothesis that selenium compounds present in the algae are responsible for the selenium induced glutathione peroxidation.     

Effect of sulphite on adenine nucleotides of the green alga Trebouxia

The amounts of AMP, ADP and ATP were determined after sulfite incubation in photo-organotrophically cultivated cells of the green alga Trebouxia. D     

Evidence for a G-Protein Regulated Adenylate Cyclase and a Ca2/Calmodulin Controlled Phosphodiesterase in the Phytoflagellate Chlorogonium

In the green flagellate Chlorogonium elongatum the cAMP concentration of the cells depends on the culture conditions of the alga. Autotrophically cultured cells possess three times more cAMP than cells cultured heterotrophically (in the dark with acetate). The activity of adenylate cyclase in a membrane fraction of Chlorogonium is distinctly stimulated by GTPγS. Such a stimulation is generally regarded as an indication of the participation of heterotrimeric G-proteins in this regulatory process. The activity of phosphodiesterase is stimulated in vitro by calmodulin and inhibited by trifluoperazin and EGTA.     

Early Devonian (~410 mya) microfossils resembling Characiopsis (Tribophyceae) and Characium (Chlorophyceae)

Unusual microfossils that occurred associated with fungal spores in the Lower Devonian (~410 mya) Windyfield chert from Scotland were composed of a narrow stipe (2.5–9 μm long) to which was attached an obovoid or elongate drop‐shaped cell up to 14 μm long; a basal attachment pad was present in several specimens. The fossils were strikingly similar morphologically to certain present‐day unicellular freshwater Tribophyceae and Chlorophyceae, but affinities to the fungal phylum Chytridiomycota also cannot be ruled out. This discovery adds to the inventory of distinctive microbial morphologies in the early non‐marine paleoecosystems.     

Identity and physiology of a new psychrophilic eukaryotic green alga, <Emphasis Type="Italic">Chlorella</Emphasis> sp., strain BI,isolated from a transitory pond near Bratina Island,Antarctica

Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the "photopsychrophiles") in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10 degrees C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes.