Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions.

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins.

Novel molecular determinants of viral susceptibility and resistance in the lipidome of Emiliania huxleyi.

Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E. huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E. huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E. huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E. huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E. huxleyi-EhV system.

Dynamic Phycobilin Pigment Variations in Diazotrophic and Non-diazotrophic Cyanobacteria Batch Cultures Under Different Initial Nitrogen Concentrations.

Increased anthropogenic nutrient loading has led to eutrophication of aquatic ecosystems, which is the major cause of harmful cyanobacteria blooms. Element stoichiometry of cyanobacteria bloom is subject to nutrient availabilities and may significantly contribute to primary production and biogeochemical cycling. Phycobilisome is the antenna of the photosynthetic pigment apparatus in cyanobacteria, which contains phycobilin pigments (PBPs) and linker proteins. This nitrogen (N)-rich protein complex has the potential to support growth as a N-storage site and may play a major role in the variability of cyanobacteria N stoichiometry. However, the regulation of PBPs during bloom formation remains unclear. We investigated the temporal variation of N allocation into PBPs and element stoichiometry for two ubiquitous cyanobacteria species, Microcystis aeruginosa and Dolichospermum flos-aquae, in a batch culture experiment with different initial N availabilities. Our results indicated that the N allocation into PBPs is species-dependent and tightly regulated by the availability of nutrients fueling population expansion. During the batch culture experiment, different nutrient uptake rates led to distinct stoichiometric imbalances of N and phosphorus (P), which substantially altered cyanobacteria C: N and C: P stoichiometry. Microcystis invested cellular N into PBPs and exhibited greater flexibility in C: N and C: P stoichiometry than D. flos-aquae. The dynamics of such N-rich macromolecules may help explain the N stoichiometry variation during a bloom and the interspecific difference between M. aeruginosa and D. flos-aquae. Our study provides a quantitative understanding of the elemental stoichiometry and the regulation of PBPs for non-diazotrophic and diazotrophic cyanobacteria blooms.

Measurement of 13C and 15N isotopic composition on nanomolar quantities of C and N.

We describe a trapping and chromatography system that cryogenically removes CO(2) and N(2) generated from sample combustion in an elemental analyzer (EA) and introduces these gases into a low-flow helium carrier stream for isotopic analysis. The sample size required for measurement by this system (termed nano-EA/IRMS) is almost 3 orders of magnitude less than conventional EA analyses and fills an important niche in the range of analytical isotopic methods. Only 25 nmol of N and 41 nmol of C are needed to achieve 1.0 per thousand precision (2sigma) from a single measurement while larger samples and replicate measurements provide better precision. Analyses of standards demonstrate that nano-EA measurements are both accurate and precise, even on nanomolar quantities of C and N. Conventional and nano-EA measurements on international and laboratory standards are indistinguishable within analytical precision. Likewise, nano-EA values for international standards do not differ statistically from their consensus values. Both observations indicate the nano-EA measurements are comparable to conventional EA analyses and accurately reproduce the VPDB and AIR isotopic scales. Critical to the success of the nano-EA system is the procedure for removing the blank contribution to the measured values. Statistical treatment of uncertainties for this procedure yields an accurate method for calculating internal and external precision.

Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane-oxidizing archaea.

Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy-archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH3 -tetrahydromethanopterin (H4 MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in 13 C (mean δ13 C = -27‰ for Hydrate Ridge and -80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ13 C = -97‰ for Hydrate Ridge and -122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl-CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of - 73‰ to - 77‰ for F430 and - 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ13 C value for Hydrate Ridge was 13 C-enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.

Temperature-induced viral resistance in Emiliania huxleyi (Prymnesiophyceae).

Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi's susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.