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Nutrient availability is an important factor controlling phytoplankton productivity. Phytoplankton contribute c. 50% of the global photosynthesis and possess efficient acclimation mechanisms to cope with nutrient stress. We investigate the cellular response of the bloom-forming coccolithophore Emiliania huxleyi to phosphorus (P) scarcity, which is often a limiting factor in marine ecosystems. We combined mass spectrometry, fluorescence microscopy, transmission electron microscopy (TEM) and gene expression analyses in order to assess diverse cellular features in cells exposed to P limitation and recovery. Early starvation-induced substitution of phospholipids in the cells' membranes with galacto- and betaine lipids. Lipid remodeling was rapid and reversible upon P resupply. The PI3K inhibitor wortmannin reduced phospholipid substitution, suggesting a possible involvement of PI3K- signaling in this process. In addition, P limitation enhanced the formation and acidification of membrane vesicles in the cytoplasm. Intracellular vesicles may facilitate the recycling of cytoplasmic content, which is engulfed in the vesicles and delivered to the main vacuole. Long-term starvation was characterized by a profound increase in cell size and morphological alterations in cellular ultrastructure. This study provides cellular and molecular basis for future ecophysiological assessment of natural E. huxleyi populations in oligotrophic regions., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

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 software's 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 .

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.

Phosphorus in the +5 oxidation state (i.e., phosphate) is the most abundant form of phosphorus in the global ocean. An enigmatic pool of dissolved phosphonate molecules, with phosphorus in the +3 oxidation state, is also ubiquitous; however, cycling of phosphorus between oxidation states has remained poorly constrained. Using simple incubation and chromatography approaches, we measured the rate of the chemical reduction of phosphate to P(III) compounds in the western tropical North Atlantic Ocean. Colonial nitrogen-fixing cyanobacteria in surface waters played a critical role in phosphate reduction, but other classes of plankton, including potentially deep-water archaea, were also involved. These data are consistent with marine geochemical evidence and microbial genomic information, which together suggest the existence of a vast oceanic phosphorus redox cycle., (Copyright © 2015, American Association for the Advancement of Science.)

Diatoms and other phytoplankton play a crucial role in the global carbon cycle, fixing CO2 into organic carbon, which may then be exported to depth via sinking particles. The molecular diversity of this organic carbon is vast and many highly bioactive molecules have been identified. Polyunsaturated aldehydes (PUAs) are bioactive on various levels of the marine food web, and yet the potential for these molecules to affect the fate of organic carbon produced by diatoms remains an open question. In this study, the effects of PUAs on the natural microbial assemblages associated with sinking particles were investigated. Sinking particles were collected from 150 m in the water column and exposed to varying concentrations of PUAs in dark incubations over 24 h. PUA doses ranging from 1 to 10 µM stimulated respiration, organic matter hydrolysis, and cell growth by bacteria associated with sinking particles. PUA dosages near 100 µM appeared to be toxic, resulting in decreased bacterial cell abundance and metabolism, as well as pronounced shifts in bacterial community composition. Sinking particles were hot spots for PUA production that contained concentrations within the stimulatory micromolar range in contrast to previously reported picomolar concentrations of these compounds in bulk seawater. This suggests PUAs produced in situ stimulate the remineralization of phytoplankton-derived sinking organic matter, decreasing carbon export efficiency, and shoaling the average depths of nutrient regeneration. Our results are consistent with a "bioactivity hypothesis" for explaining variations in carbon export efficiency in the oceans.

About half the carbon fixed by phytoplankton in the ocean is taken up and metabolized by marine bacteria, a transfer that is mediated through the seawater dissolved organic carbon (DOC) pool. The chemical complexity of marine DOC, along with a poor understanding of which compounds form the basis of trophic interactions between bacteria and phytoplankton, have impeded efforts to identify key currencies of this carbon cycle link. Here, we used transcriptional patterns in a bacterial-diatom model system based on vitamin B12 auxotrophy as a sensitive assay for metabolite exchange between marine plankton. The most highly up-regulated genes (up to 374-fold) by a marine Roseobacter clade bacterium when cocultured with the diatom Thalassiosira pseudonana were those encoding the transport and catabolism of 2,3-dihydroxypropane-1-sulfonate (DHPS). This compound has no currently recognized role in the marine microbial food web. As the genes for DHPS catabolism have limited distribution among bacterial taxa, T. pseudonana may use this sulfonate for targeted feeding of beneficial associates. Indeed, DHPS was both a major component of the T. pseudonana cytosol and an abundant microbial metabolite in a diatom bloom in the eastern North Pacific Ocean. Moreover, transcript analysis of the North Pacific samples provided evidence of DHPS catabolism by Roseobacter populations. Other such biogeochemically important metabolites may be common in the ocean but difficult to discriminate against the complex chemical background of seawater. Bacterial transformation of this diatom-derived sulfonate represents a previously unidentified and likely sizeable link in both the marine carbon and sulfur cycles.

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.

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.

Phytoplankton blooms are ephemeral events of exceptionally high primary productivity that regulate the flux of carbon across marine food webs [1-3]. Quantification of bloom turnover [4] is limited by a fundamental difficulty to decouple between physical and biological processes as observed by ocean color satellite data. This limitation hinders the quantification of bloom demise and its regulation by biological processes [5, 6], which has important consequences on the efficiency of the biological pump of carbon to the deep ocean [7-9]. Here, we address this challenge and quantify algal blooms' turnover using a combination of satellite and in situ data, which allows identification of a relatively stable oceanic patch that is subject to little mixing with its surroundings. Using a newly developed multisatellite Lagrangian diagnostic, we decouple the contributions of physical and biological processes, allowing quantification of a complete life cycle of a mesoscale (∼10-100 km) bloom of coccolithophores in the North Atlantic, from exponential growth to its rapid demise. We estimate the amount of organic carbon produced during the bloom to be in the order of 24,000 tons, of which two-thirds were turned over within 1 week. Complimentary in situ measurements of the same patch area revealed high levels of specific viruses infecting coccolithophore cells, therefore pointing at the importance of viral infection as a possible mortality agent. Application of the newly developed satellite-based approaches opens the way for large-scale quantification of the impact of diverse environmental stresses on the fate of phytoplankton blooms and derived carbon in the ocean., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The mechanisms by which phytoplankton cope with stressors in the marine environment are neither fully characterized nor understood. As viruses are the most abundant entities in the global ocean and represent a strong top-down regulator of phytoplankton abundance and diversity, we sought to characterize the cellular response of two marine haptophytes to virus infection in order to gain more knowledge about the nature and diversity of microalgal responses to this chronic biotic stressor. We infected laboratory cultures of the haptophytes Haptolina ericina and Phaeocystis pouchetii with CeV-01B or PpV-01B dsDNA viruses, respectively, and assessed the extent to which host cellular responses resemble programmed cell death (PCD) through the activation of diagnostic molecular and biochemical markers. Pronounced DNA fragmentation and activation of cysteine aspartate-specific proteases (caspases) were only detected in virus-infected cultures of these phytoplankton. Inhibition of host caspase activity by addition of the pan-caspase inhibitor z-VAD-fmk did not impair virus production in either host-virus system, differentiating it from the Emiliania huxleyi -Coccolithovirus model of haptophyte-virus interactions. Nonetheless, our findings point to a general conservation of PCD-like activation during virus infection in ecologically diverse haptophytes, with the subtle heterogeneity of cell death biochemical responses possibly exerting differential regulation on phytoplankton abundance and diversity.

Phytoplankton alter their biochemical composition according to nutrient availability, such that their bulk elemental composition varies across oceanic provinces. However, the links between plankton biochemical composition and variation in biogeochemical cycling of nutrients remain largely unknown. In a survey of phytoplankton phosphorus stress in the western North Atlantic, we found that phytoplankton in the phosphorus-depleted subtropical Sargasso Sea were enriched in the biochemical polyphosphate (polyP) compared with nutrient-rich temperate waters, contradicting the canonical oceanographic view of polyP as a luxury phosphorus storage molecule. The enrichment in polyP coincided with enhanced alkaline phosphatase activity and substitution of sulfolipids for phospholipids, which are both indicators of phosphorus stress. Further, polyP appeared to be liberated preferentially over bulk phosphorus from sinking particles in the Sargasso Sea, thereby retaining phosphorus in shallow waters. Thus, polyP cycling may form a feedback loop that attenuates the export of phosphorus when it becomes scarce, contributes bioavailable P for primary production, and supports the export of carbon and nitrogen via sinking particles.

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., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

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.

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4',6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference.

Quantitative information regarding the endmember composition of the gas and oil that flowed from the Macondo well during the Deepwater Horizon oil spill is essential for determining the oil flow rate, total oil volume released, and trajectories and fates of hydrocarbon components in the marine environment. Using isobaric gas-tight samplers, we collected discrete samples directly above the Macondo well on June 21, 2010, and analyzed the gas and oil. We found that the fluids flowing from the Macondo well had a gas-to-oil ratio of 1,600 standard cubic feet per petroleum barrel. Based on the measured endmember gas-to-oil ratio and the Federally estimated net liquid oil release of 4.1 million barrels, the total amount of C(1)-C(5) hydrocarbons released to the water column was 1.7 10(11) g. The endmember gas and oil compositions then enabled us to study the fractionation of petroleum hydrocarbons in discrete water samples collected in June 2010 within a southwest trending hydrocarbon-enriched plume of neutrally buoyant water at a water depth of 1,100 m. The most abundant petroleum hydrocarbons larger than C(1)-C(5) were benzene, toluene, ethylbenzene, and total xylenes at concentrations up to 78 μg L(-1). Comparison of the endmember gas and oil composition with the composition of water column samples showed that the plume was preferentially enriched with water-soluble components, indicating that aqueous dissolution played a major role in plume formation, whereas the fates of relatively insoluble petroleum components were initially controlled by other processes.

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.

Algal and plant production of nonphosphorus lipids in place of phospholipids is a physiological response to low phosphorus (P) availability. This response has been shown in culture and in marine plankton studies, but examples from freshwater algae remain minimal. Herein, we analyzed the nutrient contents and lipid composition of periphyton communities across the Florida Everglades ecosystem. We hypothesized that in phosphate-poor areas, periphyton in high- and low-sulfate waters would vary the proportion of sulfolipids (SLs) and betaine lipids (BLs), respectively. In phosphate-enriched areas, periphyton would produce more phospholipids (PLs). We observed that at low-P sites, PLs were a minor lipid component. In cyanobacteria-dominated periphyton where sulfate was abundant, BLs were only slightly more abundant than SLs. However, in the low-P, low-sulfate area, periphyton were comprised to a greater degree green algae and diatoms, and BLs represented the majority of the total lipids. Even in a P-rich area, PLs were a small component of periphyton lipid profiles. Despite the phosphorus limitations of the Everglades, periphyton can develop tremendous biomass. Our results suggest a physiological response by periphyton to oligotrophic conditions whereby periphyton increase abundances of nonphosphorus lipids and have reduced proportions of PLs., (© 2012 Phycological Society of America.)

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO(2) and N(2), have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.

A central component of the ocean's biological carbon pump is the export of sinking, photosynthetically derived, particulate organic carbon (POC). Bacteria colonize these particles and produce enzymes that hydrolyse sinking POC thereby acting as one of the major controls on the biological pump. Here we provide evidence that a bacterial cell-cell communication mechanism, quorum sensing (QS), may influence the activity of hydrolytic enzymes on sinking particles. We collected sinking POC from a site off Vancouver Island, Canada and found that it contained acylated homoserine lactones (AHLs), a suite of well-known bacterial communication molecules. Furthermore, we observed that the addition of exogenous AHLs to incubations containing sinking POC affected the activity of key hydrolytic enzymes involved in POC degradation in some cases. Our results suggest that AHL-based QS could play an important role in regulating the degradation of sinking POC and that variability in AHL-triggered POC hydrolysis is a heretofore unrecognized process that impacts the marine biological carbon pump., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In low-phosphorus (P) marine systems, phytoplankton replace membrane phospholipids with non-phosphorus lipids, but it is not known how rapidly this substitution occurs. Here, when cells of the model diatom Thalassiosira pseudonana were transferred from P-replete medium to P-free medium, the phospholipid content of the cells rapidly declined within 48 h from 45±0.9 to 21±4.5% of the total membrane lipids; the difference was made up by non-phosphorus lipids. Conversely, when P-limited T. pseudonana were resupplied with P, cells reduced the percentage of their total membrane lipids contributed by a non-phosphorus lipid from 43±1.5 to 7.3±0.9% within 24 h, whereas the contribution by phospholipids rose from 2.2±0.1 to 44±3%. This dynamic phospholipid reservoir contained sufficient P to synthesize multiple haploid genomes, suggesting that phospholipid turnover could be an important P source for cells. Field observations of phytoplankton lipid content may thus reflect short-term changes in P supply and cellular physiology, rather than simply long-term adjustment to the environment.

Shotgun mass spectrometry was used to detect proteins in the harmful alga, Aureococcus anophagefferens, and monitor their relative abundance across nutrient replete (control), phosphate-deficient (-P) and -P refed with phosphate (P-refed) conditions. Spectral counting techniques identified differentially abundant proteins and demonstrated that under phosphate deficiency, A. anophagefferens increases proteins involved in both inorganic and organic phosphorus (P) scavenging, including a phosphate transporter, 5'-nucleotidase, and alkaline phosphatase. Additionally, an increase in abundance of a sulfolipid biosynthesis protein was detected in -P and P-refed conditions. Analysis of the polar membrane lipids showed that cellular concentrations of the sulfolipid sulphoquinovosyldiacylglycerol (SQDG) were nearly two-fold greater in the -P condition versus the control condition, while cellular phospholipids were approximately 8-fold less. Transcript and protein abundances were more tightly coupled for gene products involved in P metabolism compared to those involved in a range of other metabolic functions. Comparison of protein abundances between the -P and P-refed conditions identified differences in the timing of protein degradation and turnover. This suggests that culture studies examining nutrient starvation responses will be valuable in interpreting protein abundance patterns for cellular nutritional status and history in metaproteomic datasets.

The Deepwater Horizon blowout is the largest offshore oil spill in history. We present results from a subsurface hydrocarbon survey using an autonomous underwater vehicle and a ship-cabled sampler. Our findings indicate the presence of a continuous plume of oil, more than 35 kilometers in length, at approximately 1100 meters depth that persisted for months without substantial biodegradation. Samples collected from within the plume reveal monoaromatic petroleum hydrocarbon concentrations in excess of 50 micrograms per liter. These data indicate that monoaromatic input to this plume was at least 5500 kilograms per day, which is more than double the total source rate of all natural seeps of the monoaromatic petroleum hydrocarbons in the northern Gulf of Mexico. Dissolved oxygen concentrations suggest that microbial respiration rates within the plume were not appreciably more than 1 micromolar oxygen per day.

Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of (33)P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with (33)P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using (3)H-leucine and (3)H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga-like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of (3)H-leucine or (3)H-thymidine., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

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.

There is growing evidence that dissolved phosphorus can regulate planktonic production in the oceans' subtropical gyres, yet there is little quantitative information about the biochemical fate of phosphorus in planktonic communities. We observed in the North Pacific Subtropical Gyre (NPSG) that the synthesis of membrane lipids accounted for 18-28% of the phosphate (PO4(3-)) taken up by the total planktonic community. Paradoxically, Prochlorococcus, the cyanobacterium that dominates NPSG phytoplankton, primarily synthesizes sulfoquinovosyldiacylglycerol (SQDG), a lipid that contains sulfur and sugar instead of phosphate. In axenic cultures of Prochlorococcus, it was observed that <1% of the total PO4(3-) uptake was incorporated into membrane lipids. Liquid chromatography/mass spectrometry of planktonic lipids in the NPSG confirmed that SQDG was the dominant membrane lipid. Furthermore, the analyses of SQDG synthesis genes from the Sargasso Sea environmental genome showed that the use of sulfolipids in subtropical gyres was confined primarily to picocyanobacteria; no sequences related to known heterotrophic bacterial SQDG lineages were found. This biochemical adaptation by Prochlorococcus must be a significant benefit to these organisms, which compete against phospholipid-rich heterotrophic bacteria for PO4(3-). Thus, evolution of this "sulfur-for-phosphorus" strategy set the stage for the success of picocyanobacteria in oligotrophic environments and may have been a major event in Earth's early history when the relative availability of sulfate and PO4(3-) were significantly different from today's ocean.

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.