Probing the Bioavailability of Dissolved Iron to Marine Eukaryotic Phytoplankton Using In Situ Single Cell Iron Quotas.

We present a new approach for quantifying the bioavailability of dissolved iron (dFe) to oceanic phytoplankton. Bioavailability is defined using an uptake rate constant (kin-app) computed by combining data on: (a) Fe content of individual in situ phytoplankton cells; (b) concurrently determined seawater dFe concentrations; and (c) growth rates estimated from the PISCES model. We examined 930 phytoplankton cells, collected between 2002 and 2016 from 45 surface stations during 11 research cruises. This approach is only valid for cells that have upregulated their high-affinity Fe uptake system, so data were screened, yielding 560 single cell k in-app values from 31 low-Fe stations. We normalized k in-app to cell surface area (S.A.) to account for cell-size differences. The resulting bioavailability proxy (k in-app/S.A.) varies among cells, but all values are within bioavailability limits predicted from defined Fe complexes. In situ dFe bioavailability is higher than model Fe-siderophore complexes and often approaches that of highly available inorganic Fe'. Station averaged k in-app/S.A. are also variable but show no systematic changes across location, temperature, dFe, and phytoplankton taxa. Given the relative consistency of k in-app/S.A. among stations (ca. five-fold variation), we computed a grand-averaged dFe availability, which upon normalization to cell carbon (C) yields k in-app/C of 42,200 ± 11,000 L mol C-1 d-1. We utilize k in-app/C to calculate dFe uptake rates and residence times in low Fe oceanic regions. Finally, we demonstrate the applicability of k in-app/C for constraining Fe uptake rates in earth system models, such as those predicting climate mediated changes in net primary production in the Fe-limited Equatorial Pacific.

Enhanced ferrihydrite dissolution by a unicellular, planktonic cyanobacterium: a biological contribution to particulate iron bioavailability.

Iron (Fe) bioavailability, as determined by its sources, sinks, solubility and speciation, places severe environmental constraints on microorganisms in aquatic environments. Cyanobacteria are a widespread group of aquatic, photosynthetic microorganisms with especially high iron requirements. While iron exists predominantly in particulate form, little is known about its bioavailability to cyanobacteria. Some cyanobacteria secrete iron solubilizing ligands called siderophores, yet many environmentally relevant strains do not have this ability. This work explores the bioavailability of amorphous synthetic Fe-oxides (ferrihydrite) to the non-siderophore producing, unicellular cyanobacterium, Synechocystis sp PCC 6803. Iron uptake assays with 55 ferrihydrite established dissolution as a critical prerequisite for iron transport. Dissolution assays with the iron binding ligand, desferrioxamine B, demonstrated that Synechocystis 6803 enhances ferrihydrite dissolution, exerting siderophore-independent biological influence on ferrihydrite bioavailability. Dissolution mechanisms were studied using a range of experimental conditions; both cell-particle physical proximity and cellular electron flow were shown to be important determinants of bio-dissolution by Synechocystis 6803. Finally, the effects of ferrihydrite stability on bio-dissolution rates and cell physiology were measured, integrating biological and chemical aspects of ferrihydrite bioavailability. Collectively, these findings demonstrate that Synechocystis 6803 actively dissolves ferrihydrite, highlighting a significant biological component to mineral phase iron bioavailability in aquatic environments.

Sulfur isotope homogeneity of oceanic DMSP and DMS.

Oceanic emissions of volatile dimethyl sulfide (DMS) represent the largest natural source of biogenic sulfur to the global atmosphere, where it mediates aerosol dynamics. To constrain the contribution of oceanic DMS to aerosols we established the sulfur isotope ratios ((34)S/(32)S ratio, δ(34)S) of DMS and its precursor, dimethylsulfoniopropionate (DMSP), in a range of marine environments. In view of the low oceanic concentrations of DMS/P, we applied a unique method for the analysis of δ(34)S at the picomole level in individual compounds. Surface water DMSP collected from six different ocean provinces revealed a remarkable consistency in δ(34)S values ranging between +18.9 and +20.3‰. Sulfur isotope composition of DMS analyzed in freshly collected seawater was similar to δ(34)S of DMSP, showing that the in situ fractionation between these species is small (<+1‰). Based on volatilization experiments, emission of DMS to the atmosphere results in a relatively small fractionation (-0.5 ± 0.2‰) compared with the seawater DMS pool. Because δ(34)S values of oceanic DMS closely reflect that of DMSP, we conclude that the homogenous δ(34)S of DMSP at the ocean surface represents the δ(34)S of DMS emitted to the atmosphere, within +1‰. The δ(34)S of oceanic DMS flux to the atmosphere is thus relatively constant and distinct from anthropogenic sources of atmospheric sulfate, thereby enabling estimation of the DMS contribution to aerosols.

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.

Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Better together? Lessons on sociality from Trichodesmium.

The N2-fixing cyanobacterium Trichodesmium is an important player in the oceanic nitrogen and carbon cycles. Trichodesmium occurs both as single trichomes and as colonies containing hundreds of trichomes. In this review, we explore the benefits and disadvantages of colony formation, considering physical, chemical, and biological effects from nanometer to kilometer scale. Showing that all major life challenges are affected by colony formation, we claim that Trichodesmium's ecological success is tightly linked to its colonial lifestyle. Microbial interactions in the microbiome, chemical gradients within the colony, interactions with particles, and elevated mobility in the water column shape a highly dynamic microenvironment. We postulate that these dynamics are key to the resilience of Trichodesmium and other colony formers in our changing environment.

Taxonomic distribution of metabolic functions in bacteria associated with Trichodesmium consortia.

IMPORTANCE: Colonies of the cyanobacteria Trichodesmium act as a biological hotspot for the usage and recycling of key resources such as C, N, P, and Fe within an otherwise oligotrophic environment. While Trichodesmium colonies are known to interact and support a unique community of algae and particle-associated microbes, our understanding of the taxa that populate these colonies and the gene functions they encode is still limited. Characterizing the taxa and adaptive strategies that influence consortium physiology and its concomitant biogeochemistry is critical in a future ocean predicted to have increasingly resource-depleted regions.

Iron transport in cyanobacteria - from molecules to communities.

Iron is an essential micronutrient for the ecologically important photoautotrophic cyanobacteria which are found across diverse aquatic environments. Low concentrations and poor bioavailability of certain iron species exert a strong control on cyanobacterial growth, affecting ecosystem structure and biogeochemical cycling. Here, we review the iron-acquisition pathways cyanobacteria utilize for overcoming these challenges. As the molecular details of cyanobacterial iron transport are being uncovered, an overall scheme of how cyanobacteria handle and exploit this scarce and redox-active micronutrient is emerging. Importantly, the range of biological solutions used by cyanobacteria to increase iron fluxes goes beyond transport and includes behavioral traits of colonial cyanobacteria and intricate cyanobacteria-bacteria interactions.

Colonies of the marine cyanobacterium Trichodesmium optimize dust utilization by selective collection and retention of nutrient-rich particles.

Trichodesmium, a globally important, N2-fixing, and colony-forming cyanobacterium, employs multiple pathways for acquiring nutrients from air-borne dust, including active dust collection. Once concentrated within the colony core, dust can supply Trichodesmium with nutrients. Recently, we reported a selectivity in particle collection enabling Trichodesmium to center iron-rich minerals and optimize its nutrient utilization. In this follow-up study we examined if colonies select Phosphorus (P) minerals. We incubated 1,200 Trichodesmium colonies from the Red Sea with P-free CaCO3, P-coated CaCO3, and dust, over an entire bloom season. These colonies preferably interacted, centered, and retained P-coated CaCO3 compared with P-free CaCO3. In both studies, Trichodesmium clearly favored dust over all other particles tested, whereas nutrient-free particles were barely collected or retained, indicating that the colonies sense the particle composition and preferably collect nutrient-rich particles. This unique ability contributes to Trichodesmium's current ecological success and may assist it to flourish in future warmer oceans.

Metagenomes of Red Sea Subpopulations Challenge the Use of Marker Genes and Morphology to Assess Trichodesmium Diversity.

Trichodesmium are filamentous cyanobacteria of key interest due to their ability to fix carbon and nitrogen within an oligotrophic marine environment. Their blooms consist of a dynamic assemblage of subpopulations and colony morphologies that are hypothesized to occupy unique niches. Here, we assessed the poorly studied diversity of Trichodesmium in the Red Sea, based on metagenome-assembled genomes (MAGs) and hetR gene-based phylotyping. We assembled four non-redundant MAGs from morphologically distinct Trichodesmium colonies (tufts, dense and thin puffs). Trichodesmium thiebautii (puffs) and Trichodesmium erythraeum (tufts) were the dominant species within these morphotypes. While subspecies diversity is present for both T. thiebautii and T. erythraeum, a single T. thiebautii genotype comprised both thin and dense puff morphotypes, and we hypothesize that this phenotypic variation is likely attributed to gene regulation. Additionally, we found the rare non-diazotrophic clade IV and V genotypes, related to Trichodesmium nobis and Trichodesmium miru, respectively that likely occurred as single filaments. The hetR gene phylogeny further indicated that the genotype in clade IV could represent the species Trichodesmium contortum. Importantly, we show the presence of hetR paralogs in Trichodesmium, where two copies of the hetR gene were present within T. thiebautii genomes. This may lead to the overestimation of Trichodesmium diversity as one of the copies misidentified T. thiebautii as Trichodesmium aureum. Taken together, our results highlight the importance of re-assessing Trichodesmium taxonomy while showing the ability of genomics to capture the complex diversity and distribution of Trichodesmium populations.

The role of reduction in iron uptake processes in a unicellular, planktonic cyanobacterium.

In many aquatic environments the essential micronutrient iron is predominantly complexed by a heterogeneous pool of strong organic chelators. Research on iron uptake mechanisms of cyanobacteria inhabiting these environments has focused on endogenous siderophore production and internalization. However, as many cyanobacterial species do not produce siderophores, alternative Fe acquisition mechanisms must exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, non-siderophore producing strain Synechocystis sp. PCC 6803. By applying trace metal clean techniques and a chemically controlled growth medium we obtained reliable and reproducible short-term (radioactive assays) and long-term (growth experiments) iron uptake rates. We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. Measurements of iron reduction rates and the inhibitory effect of ferrozine on growth supported this conclusion. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains.

Insights into the bioavailability of oceanic dissolved Fe from phytoplankton uptake kinetics.

Phytoplankton growth in large parts of the world ocean is limited by low availability of dissolved iron (dFe), restricting oceanic uptake of atmospheric CO2. The bioavailability of dFe in seawater is however difficult to appraise since it is bound by a variety of poorly characterized organic ligands. Here, we propose a new approach for evaluating seawater dFe bioavailability based on its uptake rate constant by Fe-limited cultured phytoplankton. We utilized seven phytoplankton species of diverse classes, sizes, and provenances to probe for dFe bioavailability in 12 seawater samples from several ocean basins and depths. All tested phytoplankton acquired organically bound Fe in any given sample at similar rates (after normalizing to cellular surface area), confirming that multiple, Fe-limited phytoplankton species can be used to probe dFe bioavailability in seawater. These phytoplankton-based uptake rate constants allowed us to compare water types, and obtain a grand average estimate of seawater dFe bioavailability. Among water types, dFe bioavailability varied by approximately four-fold, and did not clearly correlate with Fe concentrations or any of the measured Fe speciation parameters. Compared with well-studied Fe complexes, seawater dFe is more available than model siderophore Fe, but less available than inorganic Fe. Exposure of seawater to sunlight, however, significantly enhanced dFe bioavailability. The rate constants established in this work, not only facilitate comparison between water types, but also allow calculation of Fe uptake rates by phytoplankton in the ocean based on measured dFe concentrations. The approach established and verified in this study, opens a new way for determining dFe bioavailability in samples across the ocean, and enables modeling of in situ Fe uptake rates by phytoplankton using dFe concentrations from GEOTRACES datasets.

Selective collection of iron-rich dust particles by natural Trichodesmium colonies.

Dust is an important iron (Fe) source to the ocean, but its utilization by phytoplankton is constrained by rapid sinking and slow dissolution dust-bound iron (dust-Fe). Colonies of the globally important cyanobacterium, Trichodesmium, overcome these constraints by efficient dust capturing and active dust-Fe dissolution. In this study we examined the ability of Trichodesmium colonies to maximize their Fe supply from dust by selectively collecting Fe-rich particles. Testing for selectivity in particle collection, we supplied ~600 individual colonies, collected on multiple days from the Gulf of Aqaba, with natural dust and silica minerals that were either cleaned of or coated with Fe. Using a stereoscope, we counted the number of particles retained by each colony shortly after addition and following 24 h incubation with particles, and documented translocation of particles to the colony core. We observed a strong preference for Fe-rich particles over Fe-free particles in all tested parameters. Moreover, some colonies discarded the Fe-free particles they initially collected. The preferred collection of Fe-rich particles and disposal of Fe-free particles suggest that Trichodesmium can sense Fe and selectively choose Fe-rich dust particles. This ability assists Trichodesmium obtain Fe from dust and facilitate its growth and subsequent contribution to nutrient cycling and productivity in the ocean.

Metallophores associated with Trichodesmium erythraeum colonies from the Gulf of Aqaba.

Trichodesmium is a globally important marine nitrogen fixing cyanobacteria which forms colonies and utilizes atmospherically derived dust as a source for the limiting micro-nutrient iron. Here we report the identification of metallophores isolated from incubations of natural Trichodesmium colonies collected from the Gulf of Aqaba in the Red Sea. Three of our compounds were identified as the ferrioxamine siderophores B, E, and G. The remaining fifteen metallophores had mass to charge ratios that, to our knowledge, are not common to known siderophores. Putative sum formulas suggest most of these compounds were not structurally related to each other. We also found that the novel metallophores readily formed complexes with aluminium and were less specific for iron than the ferrioxamines. In our incubations of Trichodesmium colonies, the abundance of ten of the novel metallophores positively correlated with Trichodesmium biomass, but not with bacterial biomass, whilst ferrioxamine siderophores were more strongly associated with bacterial biomass. We identified ferrioxamines and our novel metallophores in filtered surface seawater samples from the Gulf of Aqaba. However, our novel metallophores were only observed in the surface seawater sample collected at the time of highest Trichodesmium abundance, while ferrioxamines were observed even when Trichodesmium was not present. We hypothesize that the novel metallophores were specifically associated with Trichodesmium colonies. Together with the bacterially produced ferrioxamines they likely contribute to a distinctive "ligandosphere" surrounding the Trichodesmium colonies, with potential implications for metal homeostasis within the colony environment.

Colonies of marine cyanobacteria Trichodesmium interact with associated bacteria to acquire iron from dust.

Iron (Fe) bioavailability limits phytoplankton growth in vast ocean regions. Iron-rich dust uplifted from deserts is transported in the atmosphere and deposited on the ocean surface. However, this dust is a poor source of iron for most phytoplankton since dust-bound Fe is poorly soluble in seawater and dust rapidly sinks out of the photic zone. An exception is Trichodesmium, a globally important, N2 fixing, colony forming, cyanobacterium, which efficiently captures and shuffles dust to its colony core. Trichodesmium and bacteria that reside within its colonies carry out diverse metabolic interactions. Here we show evidence for mutualistic interactions between Trichodesmium and associated bacteria for utilization of iron from dust, where bacteria promote dust dissolution by producing Fe-complexing molecules (siderophores) and Trichodesmium provides dust and optimal physical settings for dissolution and uptake. Our results demonstrate how intricate relationships between producers and consumers can influence productivity in the nutrient starved open ocean.

Hydrogen Dynamics in Trichodesmium Colonies and Their Potential Role in Mineral Iron Acquisition.

N2-fixing cyanobacteria mediate H2 fluxes through the opposing processes of H2 evolution, which is a by-product of the N2 fixation reaction, and H2 uptake, which is driven by uptake hydrogenases. Here, we used microelectrodes to characterize H2 and O2 dynamics in single natural colonies of the globally important N2 fixer Trichodesmium collected from the Gulf of Eilat. We observed gradually changing H2 dynamics over the course of the day, including both net H2 evolution and net H2 uptake, as well as large differences in H2 fluxes between individual colonies. Net H2 uptake was observed in colonies amended with H2 in both light and dark. Net H2 evolution was recorded in the light only, reflecting light-dependent N2 fixation coupled to H2 evolution. Both net H2 evolution and H2 uptake rates were higher before 2 pm than later in the day. These pronounced H2 dynamics in the morning coincided with strong net O2 uptake and the previously reported diel peak in N2 fixation. Later in the afternoon, when photosynthesis rates determined by O2 measurements were highest, and N2 fixation rates decrease according to previous studies, the H2 dynamics were also less pronounced. Thus, the observed diel variations in H2 dynamics reflect diel changes in the rates of O2 consumption and N2 fixation. Remarkably, the presence of H2 strongly stimulated the uptake of mineral iron by natural colonies. The magnitude of this effect was dependent on the time of day, with the strongest response in incubations that started before 2 pm, i.e., the period that covered the time of highest uptake hydrogenase activity. Based on these findings, we propose that by providing an electron source for mineral iron reduction in N2-fixing cells, H2 may contribute to iron uptake in Trichodesmium colonies.

A role for mrgA, a DPS family protein, in the internal transport of Fe in the cyanobacterium Synechocystis sp. PCC6803.

The mrgA protein of the cyanobacterium Synechocystis sp. PCC6803 is a member of the DPS Fe storage protein family. The physiological role of this protein was studied using a disruption mutant in the mrgA gene (slr1894) and by measuring intracellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that the deletion of the mrgA gene did not impair the Fe storage capacity, as the intracellular Fe quotas of the DeltamrgA cells were comparable to those of the wild type. Furthermore, the cellular response to decreasing external Fe concentrations, as detected by the emergence of the CP43' 77K fluorescence band, was similar in wild type and mutant cultures. On the other hand, a considerable slow down in the growth rate of DeltamrgA cultures was observed upon transfer from Fe replete to Fe depleted medium, indicating impeded utilization of the plentiful intracellular Fe. Based on these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth.

Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands.

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dps(tery)) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dps(tery) at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dps(tery) and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dps(tery) brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.

Iron-Nutrient Interactions within Phytoplankton.

Iron limits photosynthetic activity in up to one third of the world's oceans and in many fresh water environments. When studying the effects of Fe limitation on phytoplankton or their adaptation to low Fe environments, we must take into account the numerous cellular processes within which this micronutrient plays a central role. Due to its flexible redox chemistry, Fe is indispensable in enzymatic catalysis and electron transfer reactions and is therefore closely linked to the acquisition, assimilation and utilization of essential resources. Iron limitation will therefore influence a wide range of metabolic pathways within phytoplankton, most prominently photosynthesis. In this review, we map out four well-studied interactions between Fe and essential resources: nitrogen, manganese, copper and light. Data was compiled from both field and laboratory studies to shed light on larger scale questions such as the connection between metabolic pathways and ambient iron levels and the biogeographical distribution of phytoplankton species.

Rapid Hydrogen Peroxide release from the coral Stylophora pistillata during feeding and in response to chemical and physical stimuli.

Corals make use of different chemical compounds during interactions with prey, predators and aggressors. Hydrogen Peroxide (H2O2) is produced and released by a wide range of organisms as part of their defense against grazers or pathogens. In coral reefs, the large fluxes and relatively long half-life of H2O2, make it a potentially important info-chemical or defense molecule. Here we describe a previously unstudied phenomenon of rapid H2O2 release from the reef-building coral Stylophora pistillata during feeding on zooplankton and in response to chemical and physical stimuli. Following stimuli, both symbiotic and bleached corals were found to rapidly release H2O2 to the surrounding water for a short period of time (few minutes). The H2O2 release was restricted to the site of stimulus, and an increase in physical stress and chemical stimuli concentration resulted in elevated H2O2 release. Omission of calcium (a key regulator of exocytotic processes) from the experimental medium inhibited H2O2 release. Hence we suggest that H2O2 is actively released in response to stimuli, rather than leaking passively from the coral tissue. We estimate that at the site of stimulus H2O2 can reach concentrations potentially high enough to deter predators or motile, potentially pathogenic, bacteria.