Patricia E. Thomé

Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3‐phosphate dehydrogenase (NAD+)

A gene homologous to GPD1, coding for glycerol‐3‐phosphate dehydrogenase (sn‐glycerol 3‐phosphate: NAD+ oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1Δ mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427. Copyright

Induction of glycerol synthesis and release in cultured Symbiodinium.

Background Symbiotic dinoflagellates transfer a substantial amount of their photosynthetic products to their animal hosts. This amount has been estimated to represent up to 90% of the photosynthetically fixed carbon and can satisfy in some instances the full respiratory requirements of the host. Although in several cnidarian-dinoflagellate symbioses glycerol is the primary photosynthetic product translocated to the host, the mechanism for its production and release has not been demonstrated conclusively. Principal Findings Using Symbiodinium cells in culture we were able to reproduce the synthesis and release of glycerol in vitro by employing an inductor for glycerol synthesis, osmotic up-shocks. Photosynthetic parameters and fluorescence analysis of photosystem II showed that the inductive conditions did not have a negative effect on photosynthetic performance, suggesting that the capacity for carbon fixation by the cells was not compromised. The demand for glycerol production required to attain osmotic balance increased the expression of ribulose 1,5-bisphosphate and of glycerol 3-phosphate dehydrogenase, possibly competing with the flux of fixed carbon necessary for protein synthesis. In longer exposures of cultured Symbiodinium cells to high osmolarity, the response was analogous to photoacclimation, reducing the excitation pressure over photosystem II, suggesting that Symbiodinium cells perceived the stress as an increase in light. The induced synthesis of glycerol resulted in a reduction of growth rates. Conclusions Our results favor a hypothetical mechanism of a signaling event involving a pressure sensor that may induce the flux of carbon (glycerol) from the symbiotic algae to the animal host, and strongly suggest that carbon limitation may be a key factor modulating the population of symbionts within the host.

Cell wall involvement in the glycerol response to high osmolarity in the halotolerant yeast Debaryomyces hansenii

Osmotic stress was studied through the induction of the gene coding for glycerol 3-phosphate dehydrogenase (DhGPD1) in the halotolerant yeast Debaryomyces hansenii. This yeast responded to modifications in turgor pressure by stimulating the transcription of DhGPD1 when exposed to solutes that cause turgor stress (NaCl or sorbitol), but did not respond to water stress mediated by ethanol. In contrast to what has been documented to occur in Saccharomyces cerevisiae, D. hansenii protoplasts did not show induction in the transcription of DhGPD1 showing a limitation in their response to solute stress. The results presented indicate that the presence of the cell wall is of significance for the induction of DhGPD1 and hence for osmotic regulation in halotolerant D. hansenii. It appears that the main osmosensor that links high osmolarity with glycerol accumulation may be of a different nature in this yeast.

The PsbO homolog from Symbiodinium kawagutii (Dinophyceae) characterized using biochemical and molecular methods

A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodiniumkawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.

Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Δ). The expression of the DhGPD1 gene in the gpd1Δ background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

Indomethacin reproducibly induces metamorphosis in Cassiopea xamachana scyphistomae

Cassiopea xamachana jellyfish are an attractive model system to study metamorphosis and/or cnidarian–dinoflagellate symbiosis due to the ease of cultivation of their planula larvae and scyphistomae through their asexual cycle, in which the latter can bud new larvae and continue the cycle without differentiation into ephyrae. Then, a subsequent induction of metamorphosis and full differentiation into ephyrae is believed to occur when the symbionts are acquired by the scyphistomae. Although strobilation induction and differentiation into ephyrae can be accomplished in various ways, a controlled, reproducible metamorphosis induction has not been reported. Such controlled metamorphosis induction is necessary for an ensured synchronicity and reproducibility of biological, biochemical, and molecular analyses. For this purpose, we tested if differentiation could be pharmacologically stimulated as in Aurelia aurita, by the metamorphic inducers thyroxine, KI, NaI, Lugol’s iodine, H2O2, indomethacin, or retinol. We found reproducibly induced strobilation by 50 μM indomethacin after six days of exposure, and 10–25 μM after 7 days. Strobilation under optimal conditions reached 80–100% with subsequent ephyrae release after exposure. Thyroxine yielded inconsistent results as it caused strobilation occasionally, while all other chemicals had no effect. Thus, indomethacin can be used as a convenient tool for assessment of biological phenomena through a controlled metamorphic process in C. xamachana scyphistomae.

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4Δ, restoring the specific activity of the enzyme (a 3‐deoxy‐D‐arabino‐heptulosonate‐7‐phosphate synthase) to wild‐type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected. Copyright

Upside-down but headed in the right direction: Review of the highly versatile Cassiopea xamachana system

The upside-down jellyfish Cassiopea xamachana (Scyphozoa: Rhizostomeae) has been predominantly studied to understand its interaction with the endosymbiotic dinoflagellate algae Symbiodinium. As an easily culturable and tractable cnidarian model, it is an attractive alternative to stony corals to understanding the mechanisms driving establishment and maintenance of symbiosis. Cassiopea is also unique in requiring the symbiont in order to complete its transition to the adult stage, thereby providing an excellent model to understand symbiosis-driven development and evolution. Recently, the Cassiopea research system has gained interest beyond symbiosis in fields related to embryology, climate ecology, behavior, and more. With these developments, resources including genomes, transcriptomes, and laboratory protocols are steadily increasing. This review provides an overview of the broad range of interdisciplinary research that has utilized the Cassiopea model and highlights the advantages of using the model for future research. We dedicate this manuscript to Robert Trench, who inspired many of us to begin working in Cassiopea

Microbial composition of biofilms associated with lithifying rubble of Acropora palmata branches.

Coral reefs are among the most productive ecosystems on the planet, but are rapidly declining due to global-warming-mediated changes in the oceans. Particularly for the Caribbean region, Acropora sp. stony corals have lost ∼80% of their original coverage, resulting in vast extensions of dead coral rubble. We analyzed the microbial composition of biofilms that colonize and lithify dead Acropora palmata rubble in the Mexican Caribbean and identified the microbial assemblages that can persist under scenarios of global change, including high temperature and low pH. Lithifying biofilms have a mineral composition that includes aragonite and magnesium calcite (16 mole% MgCO(3)) and calcite, while the mineral phase corresponding to coral skeleton is basically aragonite. Microbial composition of the lithifying biofilms are different in comparison to surrounding biotopes, including a microbial mat, water column, sediments and live A. palmata microbiome. Significant shifts in biofilm composition were detected in samples incubated in mesocosms. The combined effect of low pH and increased temperature showed a strong effect after two-week incubations for biofilm composition. Findings suggest that lithifying biofilms could remain as a secondary structure on reef rubble possibly impacting the functional role of coral reefs.

Subcellular level variation in protein accumulation of the halophylic yeast Debaryomyces hansenii grown under conditions of high osmolarity.

We have analyzed electrophoretic profiles of polypeptides extracted from various cell compartments of the yeast Debaryomyces hansenii, cultured under high osmolarity and under control conditions. We tested the effect of high concentrations of solutes with an osmotic component (sorbitol), and with osmotic and ionic components combined (NaCl or KCl). Densitometric analyses of the extracted polypeptides indicated that the stressing solutes had a differential effect on the relative concentration of total proteins as well as in proteins extracted from three subcellular compartments. Sorbitol caused a significant decrease in the concentration of various polypeptides associated with the mitochondria and the cytoplasm. By contrast, sodium ions elicited marked increases in concentration in four cytoplasmic polypeptides. KCl did not have a major effect in any of the subcellular compartments. Polypeptides were grouped as having a general osmotic response, or as having a response apparently modulated by the particular ionic environment of the growth medium. In all treatments, the number of polypeptides with an increase in their relative concentration was roughly similar to the number of polypeptides with a decrease in concentration, both relative to controls. Our results agree with previous observations on the complexity of the osmoregulatory response involving proteins whose concentration depends on the solute causing the stress. The results also indicate that subcellular compartments respond differently to stressors.