Halogenation-Dependent Effects of the Chlorosulfolipids of Ochromonas danica on Lipid Bilayers.

The chlorosulfolipids are amphiphilic natural products with stereochemically complex patterns of chlorination and sulfation. Despite their role in toxic shellfish poisoning, potential pharmacological activities, and unknown biological roles, they remain understudied due to the difficulties in purifying them from natural sources. The structure of these molecules, with a charged sulfate group in the middle of the hydrophobic chain, appears incompatible with the conventional lipid bilayer structure. Questions about chlorosulfolipids remain unanswered partly due to the unavailability of structural analogues with which to conduct structure-function studies. We approach this problem by combining enantioselective total synthesis and membrane biophysics. Using a combination of Langmuir pressure-area isotherms of lipid monolayers, fluorescence imaging of vesicles, mass spectrometry imaging, natural product isolation, small-angle X-ray scattering, and cryogenic electron microscopy, we show that danicalipin A (1) likely inserts into lipid bilayers in the headgroup region and alters their structure and phase behavior. Specifically, danicalipin A (1) thins the bilayer and fluidizes it, allowing even saturated lipid to form fluid bilayers. Lipid monolayers show similar fluidizing upon insertion of danicalipin A (1). Furthermore, we show that the halogenation of the molecule is critical for its membrane activity, likely due to sterically controlled conformational changes. Synthetic unchlorinated and monochlorinated analogues do not thin and fluidize lipid bilayers to the same extent as the natural product. Overall, this study sheds light on how amphiphilic small molecules interact with lipid bilayers and the importance of stereochemistry and halogenation for this interaction.

A Noncanonical Chromophore Reveals Structural Rearrangements of the Light-Oxygen-Voltage Domain upon Photoactivation.

Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A'α helix is observed. With FMN, a "flip" of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead "swings" away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.

Identification of fishy odor causing compounds produced by Ochromonas sp. and Cryptomonas ovate with gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography.

Disgusting fishy odor problems have become a major concern in drinking water quality, and are commonly related to algal proliferation in source water. Unlike the typical musty/earthy odorants 2-methylisoborneol (MIB) and geosmin, identification of the corresponding fishy odorants is still a big challenge. In this study, two species of fishy-odor-producing algae, Ochromonas sp. and Cryptomonas ovate, were cultured to explore the odor production characteristics and typical odorants. When algae were ruptured in the stationary and decline phases, fishy odor intensities of 4 to 8 characterized by FPA were produced. However, some frequently reported aldehydes that could cause fishy odor, including n-hexanal, 2-octenal, heptanal, 2,4-heptanal and 2,4-decadienal, were not detected in either of the cultured algae. The possible fishy odor-causing compounds were further identified by combining gas chromatography-olfactometry (GC-O/MS) and comprehensive two-dimensional gas chromatography (GC × GC-TOFMS) using retention indices (RIs). From GC-O/MS analysis, twelve and six olfactometry peaks with various odor characteristics were identified in Ochromonas sp. and Cryptomonas ovate, respectively, of which three and two olfactometry peaks showed fishy odor characteristics. 2-Nonenal, 2,4-octadienal, fluorene and 2-tetradecanone were identified as fishy odorants in Ochromonas sp., and 1-octen-3-ol, 6-methyl-5-hepten-2-one, 1-octen-3-one, 2-nonenal and 2,4-octadienal were identified in Cryptomonas ovate. Other identified compounds, including butyl butanoate (fragrant odor), ionone (fragrant odor), bis (2-chloroisopropyl) ether (chemical odor) etc., did not show fishy features. Therefore, the fishy odor might be a synthetic and comprehensive odor, which resulted from the combination of different odorants and their synergistic effects. The results of this study will be helpful for understanding fishy odor problems, which will provide support for fishy odor management and control in the drinking water industry.

The Alga Ochromonas danica Produces Bromosulfolipids.

Many halogenases interchangeably incorporate chlorine and bromine into organic molecules. On the basis of an unsubstantiated report that the alga Ochromonas danica, a prodigious producer of chlorosulfolipids, was able to produce bromosulfolipids, we have investigated the promiscuity of its halogenases toward bromine incorporation. We have found that bromosulfolipids are produced with the exact positional and stereochemical selectivity as in the chlorosulfolipid danicalipin A when this alga is grown under modified conditions containing excess bromide ion.

Asymmetric total synthesis of danicalipin A and evaluation of biological activity.

Asymmetric total synthesis of danicalipin A was achieved. The synthesis was characterized by diastereoselective introduction of chlorine atoms. Biological activities with synthetic danicalipin A, its enantiomer, and racemate were also evaluated toward brine shrimp. Both enantiomers of danicalipin A showed almost the same activity.

Asymmetric total synthesis of (+)-danicalipin A.

A convergent asymmetric total synthesis of (+)-danicalipin A is accomplished, in which two chlorinated fragments are stereoselectively joined by 1,3-dipolar coupling, leading to the confirmation of the absolute configuration of the natural product.

Element content of Ochromonas danica: a replicated chemostat study controlling the growth rate and temperature.

Ecological stoichiometry focuses on the balance between multiple nutrient elements in resources and in consumers of those resources. The major consumers of bacteria in aquatic food webs are heterotrophic and mixotrophic nanoflagellates. Despite the importance of this consumer-resource interaction to understanding nutrient dynamics in the aquatic food web, few data are available addressing the element stoichiometry of flagellate consumers. Ochromonas danica, a mixotrophic bacterivore, was used as a model organism to study the relationships among temperature, growth rate and element stoichiometry. Ochromonas danica was grown in chemostats at dilution rates ranging between 0.03 and 0.10 h(-1) and temperatures ranging between 15 and 28 °C. Cells accumulated elements as interactive functions of temperature and growth rate, with the highest element concentrations corresponding to cells grown at a low temperature and high growth rates. The highest concentrations of elements were associated with small cells. Temperature and growth rate affected the element stoichiometry (as C:N, C:P and N:P) of O. danica in a complex manner, but the growth rate had a greater effect on ratios than did temperature.

Element stoichiometry of a mixotrophic protist grown under varying resource conditions.

The balance of essential elements (e.g. carbon [C], nitrogen [N], and phosphorus [P]) between consumers and their resources influences not only the growth and reproduction of the consumers but also the nutrients they regenerate. Flagellate protists are significant predators of aquatic bacteria and directly influence nutrient flow to higher trophic levels and, through excretion, influence the mineral element composition of dissolved nutrients. Because the element stoichiometry of protists is poorly characterized, we varied the resource composition of the bacterium Pseudomonas fluorescens and used it to grow the mixotrophic bacterivorous flagellate Ochromonas danica. Using a mass balance approach, the element composition of O. danica was found to vary depending upon the nutrient composition of the prey and ranged between 482:36:1 and 80:12:1 (C:N:P molar). Homeostasis plots suggested that flagellate protists weakly regulate their element composition and are likely to regenerate different elements depending upon the nature of the element limiting growth of their prey.

Antibiotic effects of three strains of chrysophytes (Ochromonas, Poterioochromonas) on freshwater bacterial isolates.

We investigated the antibiotic effects of extracts of freeze-dried biomass and culture supernatants from the mixotrophic chrysophyte species Ochromonas danica, Poterioochromonas sp. strain DS, and Poterioochromonas malhamensis on bacterial strains isolated from lake water. Methanolic biomass extracts inhibited the growth of all tested strains, albeit to a different extent, whereas aqueous biomass extracts only affected bacteria of the genus Flectobacillus. The antibiotic action of supernatants from flagellate cultures could be mostly attributed to lipophilic substances, but the growth of bacteria affiliated with Flectobacillus and Sphingobium was also affected by hydrophilic compounds. A comparison of biomass extracts from light- and dark-adapted cultures of Poterioochromonas sp. strain DS showed that the growth-inhibiting factor was unrelated to chlorophyll derivatives. Supernatants from a dark-adapted, phagotrophically grown flagellate culture had stronger antibiotic effects and affected more bacterial strains than the supernatant from a light-adapted culture. Significant growth reduction of a Flectobacillus isolate was already induced by extremely low concentrations of lipophilic extracts from these supernatants. Our results show that metabolites of the studied flagellates - either released actively or during cell lysis - may selectively affect the growth of some aquatic bacteria even in very small doses and thus potentially affect microbial community composition. Moreover, the antibiotic potential of mixotrophic chrysophytes may change with their nutritional mode.

Absolute configuration of chlorosulfolipids from the chrysophyta Ochromonas danica.

We isolated eight chlorosulfolipids (1-8) from the chrysophyta Ochromonas danica (IAM CS-2), including five new chlorosulfolipids (2-5, 8). The planar structures of all the compounds were elucidated by 1D and 2D NMR and ESI-MS/MS analyses. We determined the relative configuration of seven chlorosulfolipids (1-7), including the most commonly known chlorosulfolipid, 2,2,11,13,15,16-hexachlorodocosane-1,14-disulfate (1), by J-based configuration analysis (JBCA). The absolute configuration of each compound was determined using a modified Mosher's method after chemical degradation. 2,2,11,13,15,16-Hexachloro-14-docosanol-1-sulfate (2) was the most toxic to brine shrimp (Artemia salina) larvae (LC(50) 0.27 microg/mL). Compounds 1 and 4-8 were less toxic (LC(50) 2.2-6.9 microg/mL). Compound 3 was not toxic at 30 microg/mL.

Relative stereochemistry determination and synthesis of the major chlorosulfolipid from Ochromonas danica.

The relative stereochemistry of the major chlorosulfolipid of the chrysophyte alga Ochromonas danica, to which we have given the name "danicalipin A", is reported. The first synthesis of this lipid, via several stereospecific electrophilic additions to alkenes, serves to corroborate the stereochemical assignment made by NMR spectroscopy. The synthesis strategy described should be applicable to other chlorosulfolipids and should provide access to sufficient material for studies of the lipid's properties and function in membranes.