Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms.

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

Proteomic responses of the coccolithophore Emiliania huxleyi to zinc limitation and trace metal substitution.

Zinc concentrations in pelagic surface waters are within the range that limits growth in marine phytoplankton cultures. However, the influence of zinc on marine primary production and phytoplankton communities is not straightforward due to largely uncharacterized abilities for some phytoplankton to access zinc species that may not be universally bioavailable and substitute zinc with cobalt or cadmium. We used a quantitative proteomic approach to investigate these strategies and other responses to zinc limitation in the coccolithophore Emiliania huxleyi, a dominant species in low zinc waters. Zinc limitation resulted in the upregulation of metal transport proteins (ZIP, TroA-like) and COG0523 metallochaperones. Some proteins were uniquely sensitive to growth under replete zinc, substitution of zinc with cobalt, or enhancement of growth with cadmium, and may be useful as biomarkers of zinc stress or substitution in situ. Several proteins specifically upregulated under cobalt-supported or cadmium-enhanced growth appear to reflect stress responses, despite titration of these metals to optimal nutritive levels. Relief from zinc limitation by zinc or cadmium resulted in increased expression of a δ-carbonic anhydrase. Our inability to detect metal binding enzymes that are specifically induced under cobalt- or cadmium-supported growth suggests cambialism is important for zinc substitution in E. huxleyi.

Correction: Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation.

[This corrects the article DOI: 10.1371/journal.pgen.1006490.].

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation.

Environmental fluctuations affect distribution, growth and abundance of diatoms in nature, with iron (Fe) availability playing a central role. Studies on the response of diatoms to low Fe have either utilized continuous (24 hr) illumination or sampled a single time of day, missing any temporal dynamics. We profiled the physiology, metabolite composition, and global transcripts of the pennate diatom Phaeodactylum tricornutum during steady-state growth at low, intermediate, and high levels of dissolved Fe over light:dark cycles, to better understand fundamental aspects of genetic control of physiological acclimation to growth under Fe-limitation. We greatly expand the catalog of genes involved in the low Fe response, highlighting the importance of intracellular trafficking in Fe-limited diatoms. P. tricornutum exhibited transcriptomic hallmarks of slowed growth leading to prolonged periods of cell division/silica deposition, which could impact biogeochemical carbon sequestration in Fe-limited regions. Light harvesting and ribosome biogenesis transcripts were generally reduced under low Fe while transcript levels for genes putatively involved in the acquisition and recycling of Fe were increased. We also noted shifts in expression towards increased synthesis and catabolism of branched chain amino acids in P. tricornutum grown at low Fe whereas expression of genes involved in central core metabolism were relatively unaffected, indicating that essential cellular function is protected. Beyond the response of P. tricornutum to low Fe, we observed major coordinated shifts in transcript control of primary and intermediate metabolism over light:dark cycles which contribute to a new view of the significance of distinctive diatom pathways, such as mitochondrial glycolysis and the ornithine-urea cycle. This study provides new insight into transcriptional modulation of diatom physiology and metabolism across light:dark cycles in response to Fe availability, providing mechanistic understanding for the ability of diatoms to remain metabolically poised to respond quickly to Fe input and revealing strategies underlying their ecological success.

Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability.

Diatoms, unicellular phytoplankton that account for ∼40% of marine primary productivity, often dominate coastal and open-ocean upwelling zones. Limitation of growth and productivity by iron at low light is attributed to an elevated cellular Fe requirement for the synthesis of Fe-rich photosynthetic proteins. In the dynamic coastal environment, Fe concentrations and daily surface irradiance levels can vary by two to three orders of magnitude on short spatial and temporal scales. Although genome-wide studies are beginning to provide insight into the molecular mechanisms used by diatoms to rapidly respond to such fluxes, their functional role in mediating the Fe stress response remains uncharacterized. Here, we show, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid and enhances growth during acute Fe limitation at subsaturating light by increasing the photosynthetic efficiency of carbon fixation. Clone lines overexpressing TpDSP1 had a lower quantum requirement for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I. Cyclic electron flow is an ATP-producing pathway essential in higher plants and chlorophytes with a heretofore unappreciated role in diatoms. However, cells under replete conditions were characterized as having markedly reduced growth and photosynthetic rates at saturating light, thereby constraining the benefits afforded by overexpression. Widespread distribution of DSP-like sequences in environmental metagenomic and metatranscriptomic datasets highlights the presence and relevance of this protein in natural phytoplankton populations in diverse oceanic regimes.

A Mn-54 radiotracer study of Mn isotope solid-liquid exchange during reductive transformation of vernadite (δ-MnO2) by aqueous Mn(II).

We employed Mn-54 radiotracers to characterize the extent and dynamics of Mn atom exchange between aqueous Mn(II) and vernadite (δ-Mn(IV)O2) at pH 7.5 under anoxic conditions. Exchange of Mn atoms between the solid and liquid phase is rapid, reaching dynamic equilibrium in 2-4 days. We propose that during the initial stages of reaction, Mn atom exchange occurs through consecutive comproportionation-disproportionation reactions where interfacial electron transfer from adsorbed Mn(II) to lattice Mn(IV) generates labile Mn(III) cations that rapidly disproportionate to reform aqueous Mn(II) and solid-phase Mn(IV). Following nucleation of Mn(III)OOH phases, additional exchange likely occurs through electron transfer from aqueous Mn(II) to solid-phase Mn(III). Our results provide evidence for the fast and extensive production of transient Mn(III) species at the vernadite surface upon contact of this substrate with dissolved Mn(II). We further show that HEPES buffer is a reductant of lattice Mn(IV) in the vernadite structure in our experiments. The methods and results presented here introduce application of Mn-54 tracers as a facile tool to further investigate the formation kinetics of labile Mn(III) surface species and their impacts on Mn-oxide structure and reactivity over a range of environmentally relevant geochemical conditions.

Low CO2 results in a rearrangement of carbon metabolism to support C4 photosynthetic carbon assimilation in Thalassiosira pseudonana.

The mechanisms of carbon concentration in marine diatoms are controversial. At low CO2 , decreases in O2 evolution after inhibition of phosphoenolpyruvate carboxylases (PEPCs), and increases in PEPC transcript abundances, have been interpreted as evidence for a C4 mechanism in Thalassiosira pseudonana, but the ascertainment of which proteins are responsible for the subsequent decarboxylation and PEP regeneration steps has been elusive. We evaluated the responses of T. pseudonana to steady-state differences in CO2 availability, as well as to transient shifts to low CO2 , by integrated measurements of photosynthetic parameters, transcript abundances and quantitative proteomics. On shifts to low CO2 , two PEPC transcript abundances increased and then declined on timescales consistent with recoveries of Fv /Fm , non-photochemical quenching (NPQ) and maximum chlorophyll a-specific carbon fixation (Pmax ), but transcripts for archetypical decarboxylation enzymes phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) did not change. Of 3688 protein abundances measured, 39 were up-regulated under low CO2 , including both PEPCs and pyruvate carboxylase (PYC), whereas ME abundance did not change and PEPCK abundance declined. We propose a closed-loop biochemical model, whereby T. pseudonana produces and subsequently decarboxylates a C4 acid via PEPC2 and PYC, respectively, regenerates phosphoenolpyruvate (PEP) from pyruvate in a pyruvate phosphate dikinase-independent (but glycine decarboxylase (GDC)-dependent) manner, and recuperates photorespiratory CO2 as oxaloacetate (OAA).

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

When to say when: can excessive drinking explain silicon uptake in diatoms?

Diatoms are the single most important drivers of the oceanic silicon biogeochemical cycle. Due to their considerable promise in nanotechnology, there is tremendous interest in understanding the mechanism by which they produce their intricately and ornately decorated silica-based cell wall. Although specific proteins have been implicated in some of the key steps of silicification, the exact mechanisms are poorly understood.Silicon transporters, identified in both diatoms and silicoflagellates, are hypothesized to mediate silicon uptake. Recently, macropinocytosis, the non-specific engulfment of extracellular fluid, was proposed as a more energetically favorable uptake mechanism, which can also explain the long-observed effect of salinity on frustule morphology. We explore the bioenergetic, membrane recycling, and vacuolar volume requirements that must be satisfied for pinocytosis-mediated silicon uptake. These calculated requirements contrast starkly with existing data on diatom physiology, uptake kinetics, growth, and ultrastructure, leading us to conclude that pinocytosis cannot be the primary mechanism of silicon uptake.

The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom.

Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms.

A quantitative SMRT cell sequencing method for ribosomal amplicons.

Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.

Adsorption of arsenic with struvite and hydroxylapatite in phosphate-bearing solutions.

Arsenic sorption at above neutral pH is relevant when considering contaminant mobility in alkaline, phosphorus-bearing wastewaters, and may be viable in the presence phosphate minerals. Arsenic adsorption on struvite (MgNH4PO4 · 6H2O, MAP) and hydroxylapatite (Ca5(PO4)3OH, HAP) was evaluated at pH 8-11 from solutions with 2.7-0.125 mM phosphate and 0.05 mM As(III) or As(V). Over 7 d, As(III) removal from solution was minimal, but As(V) removal increased with pH, and was higher in the presence of MAP compared to HAP with a maximum of 74% removal in pH 11 MAP-bearing solutions. X-ray absorption fine structure spectroscopy (XAFS) analysis of solids recovered from pH 10-11 solutions revealed different mechanisms of As(V) sorption with MAP and HAP. Arsenic forms monodentate mononuclear surface complexes with MAP through the formation of a Mg-O-As bond, but is incorporated at the near-surface of HAP forming a johnbaumite-like (Ca5(AsO4)3OH) structure. Experiments using radioactive (33)P at pH 10-11 revealed faster exchange of P at the HAP surface, which could promote more facile As incorporation. Near-surface incorporated As in HAP may be less susceptible to remobilization compared to surface adsorbates formed with MAP. Overall, both MAP and HAP may sorb As at high pH in the presence of phosphate. This is relevant to the fate of As in alkaline contaminated waters in contact with phosphate mineral phases.

The unique biogeochemical signature of the marine diazotroph trichodesmium.

The elemental composition of phytoplankton can depart from canonical Redfield values under conditions of nutrient limitation or production (e.g., N fixation). Similarly, the trace metal metallome of phytoplankton may be expected to vary as a function of both ambient nutrient concentrations and the biochemical processes of the cell. Diazotrophs such as the colonial cyanobacteria Trichodesmium are likely to have unique metal signatures due to their cell physiology. We present metal (Fe, V, Zn, Ni, Mo, Mn, Cu, Cd) quotas for Trichodesmium collected from the Sargasso Sea which highlight the unique metallome of this organism. The element concentrations of bulk colonies and trichomes sections were analyzed by ICP-MS and synchrotron x-ray fluorescence, respectively. The cells were characterized by low P contents but enrichment in V, Fe, Mo, Ni, and Zn in comparison to other phytoplankton. Vanadium was the most abundant metal in Trichodesmium, and the V quota was up to fourfold higher than the corresponding Fe quota. The stoichiometry of 600C:101N:1P (mol mol(-1)) reflects P-limiting conditions. Iron and V were enriched in contiguous cells of 10 and 50% of Trichodesmium trichomes, respectively. The distribution of Ni differed from other elements, with the highest concentration in the transverse walls between attached cells. We hypothesize that the enrichments of V, Fe, Mo, and Ni are linked to the biochemical requirements for N fixation either directly through enrichment in the N-fixing enzyme nitrogenase or indirectly by the expression of enzymes responsible for the removal of reactive oxygen species. Unintentional uptake of V via P pathways may also be occurring. Overall, the cellular content of trace metals and macronutrients differs significantly from the (extended) Redfield ratio. The Trichodesmium metallome is an example of how physiology and environmental conditions can cause significant deviations from the idealized stoichiometry.

Quantification of nitrogenase in Trichodesmium IMS 101: implications for iron limitation of nitrogen fixation in the ocean.

Iron is widely thought to limit nitrogen fixation in the open, oligotrophic ocean due to the low solubility of Fe in oxic seawater and the high Fe demand for the nitrogenase holozyme. However, empirical evidence for Fe limitation of field populations of Trichodesmium based on either incubation experiments or molecular and physiological indicators has not quantitatively related Fe supply to the cellular Fe quotas for nitrogenase. Rather, the Fe required for N2 fixation has been inferred from in vivo catalytic activity. Using a pet14b expression vector, we cloned the nif H gene (encoding the Fe-protein, which contains 4Fe atoms per subunit) from Trichodesmium IMS 101, and purified the His-tagged apoprotein with which we derived a primary standard based on quantitative Western blots. Using a standard curve derived from the cloned Trichodesmium Fe apoprotein, we measured the absolute abundance of the Fe-protein in iron-replete cultures of this marine diazotroph. At peak expression, we calculate 0.04 mg nitrogenase mg(-1) C. Assuming a conservative stoichiometry of two Fe-protein subunits per MoFe protein (which contains 15 Fe atoms per subunit, or a total of 38 atoms of Fe per holozyme), we estimate 236 µmol Fe is bound to nitrogenase per mol cellular C. This estimate is about 10 times greater than the Fe previously calculated to support diazotrophic growth under these conditions. Our results suggest that under bloom conditions in the subtropical North Atlantic and North Pacific, as much as ∼2.22 and 0.06 µmol m(-3) of Fe is bound to Trichodesmium nitrogenase respectively. Such a high quota represents between ∼50% and > 100% summer-time average particulate Fe in surface waters, suggesting the importance of this taxon for the retention and biogeochemical cycling of Fe. Moderate growth (0.10 day(-1) ) towards the end of these blooms would require a vertical flux as high as ∼23 µmol Fe day(-1) m(-2) into the mixed layer.