Helen F. Fredricks

Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity

Phosphorus is an obligate requirement for the growth of all organisms; major biochemical reservoirs of phosphorus in marine plankton include nucleic acids and phospholipids. However, eukaryotic phytoplankton and cyanobacteria (that is, ‘phytoplankton’ collectively) have the ability to decrease their cellular phosphorus content when phosphorus in their environment is scarce. The biochemical mechanisms that allow phytoplankton to limit their phosphorus demand and still maintain growth are largely unknown. Here we show that phytoplankton, in regions of oligotrophic ocean where phosphate is scarce, reduce their cellular phosphorus requirements by substituting non-phosphorus membrane lipids for phospholipids. In the Sargasso Sea, where phosphate concentrations were less than 10 nmol l-1, we found that only 1.3 ± 0.6% of phosphate uptake was used for phospholipid synthesis; in contrast, in the South Pacific subtropical gyre, where phosphate was greater than 100 nmol l-1, plankton used 17 ± 6% (ref. 6). Examination of the planktonic membrane lipids at these two locations showed that classes of sulphur- and nitrogen-containing membrane lipids, which are devoid of phosphorus, were more abundant in the Sargasso Sea than in the South Pacific. Furthermore, these non-phosphorus, ‘substitute lipids’ were dominant in phosphorus-limited cultures of all of the phytoplankton species we examined. In contrast, the marine heterotrophic bacteria we examined contained no substitute lipids and only phospholipids. Thus heterotrophic bacteria, which compete with phytoplankton for nutrients in oligotrophic regions like the Sargasso Sea, appear to have a biochemical phosphorus requirement that phytoplankton avoid by using substitute lipids. Our results suggest that phospholipid substitutions are fundamental biochemical mechanisms that allow phytoplankton to maintain growth in the face of phosphorus limitation.

Viral Glycosphingolipids Induce Lytic Infection and Cell Death in Marine Phytoplankton

The Death of Cocco Emiliania huxleyi is a coccolithophore, a class of unicellular phytoplankton that forms vast blooms mediating the oceanic carbon cycle through shedding of its calcium carbonate scales. E. huxleyi is routinely infected and killed by lytic viruses that can abruptly halt a bloom. Vardi et al. (p. 861) have found that in E. huxleyi strains that are sensitive or resistant to infection, a sphingolipid-based “arms race” appears to regulate cell fate during host-virus interactions. The lipid also serves as a biomarker for active infection that may help to quantify the role and activity of viruses and virus-mediated processes in the oceans. This information will help in assessing the biogeochemical impact of these plankton species. A specific virus encodes membrane components that broadcast cell death and population demise of its coccolithophore host. Marine viruses that infect phytoplankton are recognized as a major ecological and evolutionary driving force, shaping community structure and nutrient cycling in the marine environment. Little is known about the signal transduction pathways mediating viral infection. We show that viral glycosphingolipids regulate infection of Emiliania huxleyi, a cosmopolitan coccolithophore that plays a major role in the global carbon cycle. These sphingolipids derive from an unprecedented cluster of biosynthetic genes in Coccolithovirus genomes, are synthesized de novo during lytic infection, and are enriched in virion membranes. Purified glycosphingolipids induced biochemical hallmarks of programmed cell death in an uninfected host. These lipids were detected in coccolithophore populations in the North Atlantic, which highlights their potential as biomarkers for viral infection in the oceans.

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.

Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host–virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the “arms race” between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi–EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host–virus interactions in natural systems.

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.

Molecular Ion-Independent Quantification of Polar Glycerolipid Classes in Marine Plankton Using Triple Quadrupole MS

Polar glycerolipids are a diverse family of lipid molecules that form the bulk of bacterial and eukaryotic microbial membranes. The earth and ocean sciences has a long history of using fatty acids as biomarkers for microbes, but have only recently begun to examine the intact polar lipids from which they are derived. Current analytical approaches rely on laboriously quantifying the molecular ions of each of these species independently. Thus, we saw a need for a method for quantifying polar glycerolipid classes that was: (i) selective for individual classes, (ii) inclusive of all species within a class, (iii) independent of foreknowledge of the molecular ions of the polar glycerolipid, and (iv) amenable to automated, high-throughput data analysis methods. Our new HPLC-electrospray-ionization triple-quadrupole MS (HPLC-ESI-TQMS) method can be applied to quantify the nine major classes of polar glycerolipid in planktonic communities: the phospholipids phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the glycolipids monoglycosyldiacylglycerol, diglycosyldiacylglycerol and sulfoquinovosyldiacylglycerol; and the betaine lipids diacylglyceryl trimethyl homoserine, diacylglyceryl hydroxymethyl trimethyl-β-alanine, and diacylglyceryl carboxyhydroxymethylcholine. The analyses rely on neutral loss and parent ion scan events that yield one chromatogram for each class of polar glycerolipid, simplifying downstream analysis and increasing sample throughput. The efficacy of the method was demonstrated by analyzing plankton community samples from a variety of marine environments.

SAR11 lipid renovation in response to phosphate starvation

Significance Nonphosphorus lipids produced by heterotrophic bacteria have been measured in marine ecosystems without an understanding of their origins or role. This work shows SAR11 chemoheterotrophic bacteria synthesize multiple nonphosphorus lipids in response to phosphate depletion. Because this process results in a reduced cellular P:C ratio, it impacts our understanding of ocean processes related to cellular elemental stoichiometry by showing how different environmental parameters alter P:C ratios in heterotrophs. Also, SAR11 grown with excess organophosphonate synthesized phosphorus-free lipids. This finding contrasts the contemporary view of organophosphorus utilization because organophosphate-derived phosphorus did not equally substitute for inorganic phosphate in lipids. Considering lipid phosphorus content was lower in cells using organophosphonate, phosphorus-based productivity estimates may vary as a function of phosphorus source. Phytoplankton inhabiting oligotrophic ocean gyres actively reduce their phosphorus demand by replacing polar membrane phospholipids with those lacking phosphorus. Although the synthesis of nonphosphorus lipids is well documented in some heterotrophic bacterial lineages, phosphorus-free lipid synthesis in oligotrophic marine chemoheterotrophs has not been directly demonstrated, implying they are disadvantaged in phosphate-deplete ecosystems, relative to phytoplankton. Here, we show the SAR11 clade chemoheterotroph Pelagibacter sp. str. HTCC7211 renovates membrane lipids when phosphate starved by replacing a portion of its phospholipids with monoglucosyl- and glucuronosyl-diacylglycerols and by synthesizing new ornithine lipids. Lipid profiles of cells grown with excess phosphate consisted entirely of phospholipids. Conversely, up to 40% of the total lipids were converted to nonphosphorus lipids when cells were starved for phosphate, or when growing on methylphosphonate. Cells sequentially limited by phosphate and methylphosphonate transformed >75% of their lipids to phosphorus-free analogs. During phosphate starvation, a four-gene cluster was significantly up-regulated that likely encodes the enzymes responsible for lipid renovation. These genes were found in Pelagibacterales strains isolated from a phosphate-deficient ocean gyre, but not in other strains from coastal environments, suggesting alternate lipid synthesis is a specific adaptation to phosphate scarcity. Similar gene clusters are found in the genomes of other marine α-proteobacteria, implying lipid renovation is a common strategy used by heterotrophic cells to reduce their requirement for phosphorus in oligotrophic habitats.

Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

LOBSTAHS: An Adduct-Based Lipidomics Strategy for Discovery and Identification of Oxidative Stress Biomarkers

Discovery and identification of molecular biomarkers in large LC/MS data sets requires significant automation without loss of accuracy in the compound screening and annotation process. Here, we describe a lipidomics workflow and open-source software package for high-throughput annotation and putative identification of lipid, oxidized lipid, and oxylipin biomarkers in high-mass-accuracy HPLC-MS data. Lipid and oxylipin biomarker screening through adduct hierarchy sequences, or LOBSTAHS, uses orthogonal screening criteria based on adduct ion formation patterns and other properties to identify thousands of compounds while providing the user with a confidence score for each assignment. Assignments are made from one of two customizable databases; the default databases contain 14 068 unique entries. To demonstrate the softwares functionality, we screened more than 340 000 mass spectral features from an experiment in which hydrogen peroxide was used to induce oxidative stress in the marine diatom Phaeodactylum tricornutum. LOBSTAHS putatively identified 1969 unique parent compounds in 21 869 features that survived the multistage screening process. While P. tricornutum maintained more than 92% of its core lipidome under oxidative stress, patterns in biomarker distribution and abundance indicated remodeling was both subtle and pervasive. Treatment with 150 μM H2O2 promoted statistically significant carbon-chain elongation across lipid classes, with the strongest elongation accompanying oxidation in moieties of monogalactosyldiacylglycerol, a lipid typically localized to the chloroplast. Oxidative stress also induced a pronounced reallocation of lipidome peak area to triacylglycerols. LOBSTAHS can be used with environmental or experimental data from a variety of systems and is freely available at https://github.com/vanmooylipidomics/LOBSTAHS .

Quantitative exploration of the contribution of settlement, growth, dispersal and grazing to the accumulation of natural marine biofilms on antifouling and fouling-release coatings

The accumulation of microbial biofilms on ships’ hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships’ hulls.