Dinoflagellates of the genus Symbiodinium are commonly recognized as invertebrate endosymbionts that are of central importance for the functioning of coral reef ecosystems. However, the endosymbiotic phase within Symbiodinium life history is inherently tied to a more cryptic free-living (ex hospite) phase that remains largely unexplored. Here we show that free-living Symbiodinium spp. in culture commonly form calcifying bacterial-algal communities that produce aragonitic spherulites and encase the dinoflagellates as endolithic cells. This process is driven by Symbiodinium photosynthesis but occurs only in partnership with bacteria. Our findings not only place dinoflagellates on the map of microbial-algal organomineralization processes but also point toward an endolithic phase in the Symbiodinium life history, a phenomenon that may provide new perspectives on the biology and ecology of Symbiodinium spp. and the evolutionary history of the coraldinoflagellate symbiosis.Symbiodinium | coral symbiont | microbial-algal calcification | endolithic algae | life history

Marine organisms, including seagrasses, are important sources of biologically active molecules for the treatment of human diseases. In this study, organic extracts of the marine seagrass Halophila stipulacea obtained by different polarities from leaves (L) and stems (S) (hexane [HL, HS], ethyl acetate [EL, ES], and methanol [ML, MS]) were tested for different bioactivities. The screening comprehended the cytotoxicity activity against cancer cell lines grown as a monolayer culture or as multicellular spheroids (cancer), glucose uptake in cells (diabetes), reduction of lipid content in fatty acid-overloaded liver cells (steatosis), and lipid-reducing activity in zebrafish larvae (obesity), as well as the antifouling activity against marine bacteria (microfouling) and mussel larval settlement (macrofouling). HL, EL, HS, and ES extracts showed statistically significant cytotoxicity against cancer cell lines. The extracts did not have any significant effect on glucose uptake and on the reduction of lipids in liver cells. The EL and ML extracts reduced neutral lipid contents on the larvae of zebrafish with EC50 values of 2.2 µg/mL for EL and 1.2 µg/mL for ML. For the antifouling activity, the HS and ML extracts showed a significant inhibitory effect (p < 0.05) against the settlement of Mytilus galloprovincialis plantigrade larvae. The metabolite profiling using HR-LC-MS/MS and GNPS (The Global Natural Product Social Molecular Networking) analyses identified a variety of known primary and secondary metabolites in the extracts, along with some unreported molecules. Various compounds were detected with known activities on cancer (polyphenols: Luteolin, apeginin, matairesinol), on metabolic diseases (polyphenols: cirsimarin, spiraeoside, 2,4-dihydroxyheptadec-16-ynyl acetate; amino acids: N-acetyl-L-tyrosine), or on antifouling (fatty acids: 13-decosenamide; cinnamic acids: 3-hydroxy-4-methoxycinnamic acid, alpha-cyano-4-hydroxycinnamic), which could be, in part, responsible for the observed bioactivities. In summary, this study revealed that Halophila stipulacea is a rich source of metabolites with promising activities against obesity and biofouling and suggests that this seagrass could be useful for drug discovery in the future.

Marine organisms, particularly cyanobacteria, are important resources for the production of bioactive secondary metabolites for the treatment of human diseases. In this study, a bioassay-guided approach was used to discover metabolites with lipid-reducing activity. Two chlorophyll derivatives were successfully isolated, the previously described 132-hydroxy-pheophytin a (1) and the new compound 132-hydroxy-pheofarnesin a (2). The structure elucidation of the new compound 2 was established based on one- and two-dimensional (1D and 2D) NMR spectroscopy and mass spectrometry. Compounds 1 and 2 showed significant neutral lipid-reducing activity in the zebrafish Nile red fat metabolism assay after 48 h of exposure with a half maximal effective concentration (EC50) of 8.9 ± 0.4 µM for 1 and 15.5 ± 1.3 µM for 2. Both compounds additionally reduced neutral lipid accumulation in 3T3-L1 multicellular spheroids of murine preadipocytes. Molecular profiling of mRNA expression of some target genes was evaluated for the higher potent compound 1, which indicated altered peroxisome proliferator activated receptor gamma (PPARγ) mRNA expression. Lipolysis was not affected. Different food materials (Spirulina, Chlorella, spinach, and cabbage) were evaluated for the presence of 1, and the cyanobacterium Spirulina, with GRAS (generally regarded as safe) status for human consumption, contained high amounts of 1. In summary, known and novel chlorophyll derivatives were discovered from marine cyanobacteria with relevant lipid-reducing activities, which in the future may be developed into nutraceuticals.

Portoamides are cyclic peptides produced and released by the cyanobacterial strain Phormidium sp. presumably to interfere with other organisms in their ecosystems ("allelopathy"). Portoamides were previously demonstrated to have an antiproliferative effect on human lung carcinoma cells, but the underlying mechanism of this activity has not been described. In the present work, the effects of portoamides on proliferation were examined in eight human cancer cell lines and two non-carcinogenic cell lines, and major differences in sensitivities were observed. To generate hypotheses with regard to molecular mechanisms of action, quantitative proteomics using 2D gel electrophoresis and MALDI-TOF/TOF were performed on the colon carcinoma cell line HT-29. The expression of proteins involved in energy metabolism (mitochondrial respiratory chain and pentose phosphate pathway) was found to be affected. The hypothesis of altered energy metabolism was tested in further experiments. Exposure to portoamides resulted in reduced cellular ATP content, likely due to decreased mitochondrial energy production. Mitochondrial hyperpolarization and reduced mitochondrial reductive capacity was observed in treated cells. Furthermore, alterations in the expression of peroxiredoxins (PRDX4, PRDX6) and components of proteasome subunits (PSB4, PSA6) were observed in portoamide-treated cells, but these alterations were not associated with detectable increases in oxidative stress. We conclude that the cytotoxic activity of portoamides is associated with disturbance of energy metabolism, and alterations in mitochondrial structure and function.

Esterification reactions are central to many aspects of industrial and biological chemistry. The formation of carboxyesters typically occurs through nucleophilic attack of an alcohol onto the carboxylate carbon. Under certain conditions employed in organic synthesis, the carboxylate nucleophile can be alkylated to generate esters from alkyl halides, but this reaction has only been observed transiently in enzymatic chemistry. Here, we report a carboxylate alkylating enzyme-BrtBthat catalyzes O-C bond formation between free fatty acids of varying chain length and the secondary alkyl halide moieties found in the bartolosides. Guided by this reactivity, we uncovered a variety of natural fatty acid-bartoloside esters, previously unrecognized products of the bartoloside biosynthetic gene cluster.