Iron uptake, transport and storage in marine brown algae.

Iron is a vital although biologically inaccessible trace nutrient for nearly all forms of life but "free" iron can be deleterious to cells and thus iron uptake and storage must be carefully controlled. The marine environment is particularly iron poor making mechanisms for its uptake and storage even more imperative. In this brief review we explore the known and potential iron uptake and storage pathways for the biologically and economically important marine brown macroalgae (seaweeds/kelps).

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell-1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

Distribution of dissolved iron and bacteria producing the photoactive siderophore, vibrioferrin, in waters off Southern California and Northern Baja.

Phytoplankton blooms can cause acute effects on marine ecosystems due either to their production of endogenous toxins or to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of these Harmful Algal Blooms are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable source of iron for phytoplankton which could in turn stimulate algal growth and support bloom dynamics. Here we correlate iron concentrations, phytoplankton cell counts, bacterial cell abundance, and copy numbers for a photoactive siderophore vibrioferrin biosynthesis gene in water samples taken from 2017 cruises in the Gulf of California, and the Pacific Ocean off the coast of northern Baja California as well as during a multiyear sampling at Scripps Pier in San Diego, CA. We find that bacteria producing the photoactive siderophore vibrioferrin, make up a surprisingly high percentage of total bacteria in Pacific/Gulf of California coastal waters (up to 9%). Vibroferrin's unique properties and the widespread prevalence of its bacterial producers suggest that it may contribute significantly to generating bioavailability of iron via photoredox reactions.

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus.

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Mössbauer Spectroscopic Characterization of Iron(III)-Polysaccharide Coordination Complexes: Photochemistry, Biological, and Photoresponsive Materials Implications.

While polycarboxylates and hydroxyl-acid complexes have long been known to be photoactive, simple carboxylate complexes which lack a significant LMCT band are not typically strongly photoactive. Hence, it was somewhat surprising that a series of reports demonstrated that materials synthesized from iron(III) and polysaccharides such as alginate (poly[guluronan-co-mannuronan]) or pectate (poly[galacturonan]) formed photoresponsive materials that convert from hydrogels to sols under the influence of visible light. These materials have numerous potential applications in areas such as photopatternable materials, materials for controlled drug delivery, and tissue engineering. Despite the near-identity of the functional units in the polysaccharide ligands, the reactivity of iron(III) hydrogels can depend on the configuration of some chiral centers in the sugar units and in the case of alginate the guluronate to mannuronate block composition, as well as pH. Here, using temperature- and field-dependent transmission Mössbauer spectroscopy, we show that the dominant iron compound detected for both the alginate and pectate gels displays features typical of a polymeric (Fe3+O6) system. The Mössbauer spectra of such systems are strongly dependent on temperature, field, size, and crystallinity, indicative of superparamagnetic relaxation of magnetically ordered nanoparticles. Pectate and alginate hydrogels differ in the size distribution of the iron oxyhydroxy nanoparticles, suggesting that in general smaller nanoparticles are more reactive. Potential biological implications of these results are also discussed.

Iron uptake and storage in the HAB dinoflagellate Lingulodinium polyedrum.

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive-oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to 57Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893.

Full length recombinant iron regulatory protein, Fur, has been isolated and characterized from the algal-associated marine bacterium Marinobacter algicola DG893. Under nondenaturing conditions the Fur protein behaves on size exclusion chromatography as a dimer while it is monomeric under SDS PAGE conditions. ICP-MS and fluorescence quenching experiments show that Mb-Fur binds a single metal ion (Zn, Mn, or Co) per monomer. Electrophoretic mobility shift assays were used to probe the interaction of Mb-Fur with the purported Fur box in the promoter region upstream of the vibrioferrin biosynthetic operon. Interaction of Mb-Fur with a 100 bp DNA fragment containing the Fur box in the presence of 10 µM Mn, Co or Zn(II) resulted in decreased migration of DNA on a 7.5% polyacrylamide gel. In the absence of the Fur protein or the metal, no interaction is seen. The presence of EDTA in the binding, loading or running buffers also abolished all activity demonstrating the importance of the metal in formation of the promoter-repressor complex. Based on a high degree of similarity between Mb-Fur and its homolog from Pseudomonas aeruginosa (PA) whose X-ray structure is known we developed a structural model for the former which suggested that only one of the several metal binding sites found in other Fur's would be functional. This is consistent with the single metal binding stoichiometry we observed. Since the purported metal binding site was one that has been described as "structural" rather than "functional" in PA and yet the monometallic Mb-Fur retains DNA Fur box binding ability it reopens the question of which site is which, or if different species have adapted the sites for different purposes.

Surface-bound iron: a metal ion buffer in the marine brown alga Ectocarpus siliculosus?

Although the iron uptake and storage mechanisms of terrestrial/higher plants have been well studied, the corresponding systems in marine algae have received far less attention. Studies have shown that while some species of unicellular algae utilize unique mechanisms of iron uptake, many acquire iron through the same general mechanisms as higher plants. In contrast, the iron acquisition strategies of the multicellular macroalgae remain largely unknown. This is especially surprising since many of these organisms represent important ecological and evolutionary niches in the coastal marine environment. It has been well established in both laboratory and environmentally derived samples, that a large amount of iron can be 'non-specifically' adsorbed to the surface of marine algae. While this phenomenon is widely recognized and has prompted the development of experimental protocols to eliminate its contribution to iron uptake studies, its potential biological significance as a concentrated iron source for marine algae is only now being recognized. This study used an interdisciplinary array of techniques to explore the nature of the extensive and powerful iron binding on the surface of both laboratory and environmental samples of the marine brown alga Ectocarpus siliculosus and shows that some of this surface-bound iron is eventually internalized. It is proposed that the surface-binding properties of E. siliculosus allow it to function as a quasibiological metal ion 'buffer', allowing iron uptake under the widely varying external iron concentrations found in coastal marine environments.

Borate as a synergistic anion for Marinobacter algicola ferric binding protein, FbpA: a role for boron in iron transport in marine life.

Boron in the ocean is generally considered a nonbiological element due to its relatively high concentration (0.4 mM) and depth independent concentration profile. Here we report an unexpected role for boron in the iron transport system of the marine bacterium Marinobacter algicola. Proteome analysis under varying boron concentrations revealed that the periplasmic ferric binding protein (Mb-FbpA) was among the proteins whose expression was most affected, strongly implicating the involvement of boron in iron utilization. Here we show that boron facilitates Fe(3+) sequestration by Mb-FbpA at pH 8 (oceanic pH) by acting as a synergistic anion (B(OH)4(1-)). Fe(3+) sequestration does not occur at pH 6.5 where boric acid (B(OH)3; pK(a) = 8.55) is the predominant species. Borate anion is also shown to bind to apo-Mb-FbpA with mM affinity at pH 8, consistent with the biological relevance implied from boron's oceanic concentration (0.4 mM). Borate is among those synergistic anions tested which support the strongest Fe(3+) binding to Mb-FbpA, where the range of anion dependent affinity constants is log K'(eff) = 21-22. Since the pKa of boric acid (8.55) lies near the pH of ocean water, changes in oceanic pH, as a consequence of fluctuations in atmospheric CO2, may perturb iron uptake in many marine heterotrophic bacteria due to a decrease in oceanic borate anion concentration.

Detection of photoactive siderophore biosynthetic genes in the marine environment.

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). While it has been hypothesized that the global production of siderophores by heterotrophic bacteria and some cyanobacteria constitutes the bulk of organic ligands binding iron in the ocean because stability constants of siderophores and these organic ligands are similar, and because ligand concentrations rise sharply in response to iron fertilization events, direct evidence for this proposal is lacking. This lack is due to the difficulty in characterizing these ligands due both to their extremely low concentrations and their highly heterogeneous nature. The situation for characterizing photoactive siderophores in situ is more problematic because of their expected short lifetimes in the photic zone. An alternative approach is to make use of high sensitivity molecular technology (qPCR) to search for siderophore biosynthesis genes related to the production of photoactive siderophores. In this way one can access their "biochemical potential" and utilize this information as a proxy for the presence of these siderophores in the marine environment. Here we show, using qPCR primers designed to detect biosynthetic genes for the siderophores vibrioferrin, petrobactin and aerobactin that such genes are widespread and based on their abundance, the "biochemical potential" for photoactive siderophore production is significant. Concurrently we also briefly examine the microbial biodiversity responsible for such production as a function of depth and location across a North Atlantic transect.

Synchrotron X-rays reveal the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus.

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (µXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

Atypical iron storage in marine brown algae: a multidisciplinary study of iron transport and storage in Ectocarpus siliculosus.

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood, with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins, while strategy II plants utilize high-affinity, iron-specific, binding compounds called phytosiderophores. In contrast, little is known about the corresponding systems in marine, plant-like lineages, particularly those of multicellular algae (seaweeds). Herein the first study of the iron uptake and storage mechanisms in the brown alga Ectocarpus siliculosus is reported. Genomic data suggest that Ectocarpus may use a strategy I approach. Short-term radio-iron uptake studies verified that iron is taken up by Ectocarpus in a time- and concentration-dependent manner consistent with an active transport process. Upon long-term exposure to (57)Fe, two metabolites have been identified using a combination of Mössbauer and X-ray absorption spectroscopies. These include an iron-sulphur cluster accounting for ~26% of the total intracellular iron pool and a second component with spectra typical of a polymeric (Fe(3+)O(6)) system with parameters similar to the amorphous phosphorus-rich mineral core of bacterial and plant ferritins. This iron metabolite accounts for ~74% of the cellular iron pool and suggests that Ectocarpus contains a non-ferritin but mineral-based iron storage pool.

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light.

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent "carbon for iron mutualism". While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in "algal-associated" VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal-bacterial "carbon for iron mutualism" hypothesis in the marine environment.

Iron transport in the genus Marinobacter.

Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world's oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.

Photolysis of iron-siderophore chelates promotes bacterial-algal mutualism.

Marine microalgae support world fisheries production and influence climate through various mechanisms. They are also responsible for harmful blooms that adversely impact coastal ecosystems and economies. Optimal growth and survival of many bloom-forming microalgae, including climatically important dinoflagellates and coccolithophores, requires the close association of specific bacterial species, but the reasons for these associations are unknown. Here, we report that several clades of Marinobacter ubiquitously found in close association with dinoflagellates and coccolithophores produce an unusual lower-affinity dicitrate siderophore, vibrioferrin (VF). Fe-VF chelates undergo photolysis at rates that are 10-20 times higher than siderophores produced by free-living marine bacteria, and unlike the latter, the VF photoproduct has no measurable affinity for iron. While both an algal-associated bacterium and a representative dinoflagellate partner, Scrippsiella trochoidea, used iron from Fe-VF chelates in the dark, in situ photolysis of the chelates in the presence of attenuated sunlight increased bacterial iron uptake by 70% and algal uptake by >20-fold. These results suggest that the bacteria promote algal assimilation of iron by facilitating photochemical redox cycling of this critical nutrient. Also, binary culture experiments and genomic evidence suggest that the algal cells release organic molecules that are used by the bacteria for growth. Such mutualistic sharing of iron and fixed carbon has important implications toward our understanding of the close beneficial interactions between marine bacteria and phytoplankton, and the effect of these interactions on algal blooms and climate.

Photoactive siderophores: Structure, function and biology.

It is well known that bacteria and fungi have evolved sophisticated systems for acquiring the abundant but biologically inaccessible trace element iron. These systems are based on high affinity Fe(III)-specific binding compounds called siderophores which function to acquire, transport, and process this essential metal ion. Many hundreds of siderophores are now known and their numbers continue to grow. Extensive studies of their isolation, structure, transport, and molecular genetics have been undertaken in the last three decades and have been comprehensively reviewed many times. In this review we focus on a unique subset of siderophores that has only been recognized in the last 20 years, namely those whose iron complexes display photoactivity. This photoactivity, which typically results in the photooxidation of the siderophore ligand with concomitant reduction of Fe(III) to Fe(II), seemingly upsets the siderophore paradigm of forming and transporting only extremely stable Fe(III) complexes into microbial cells. Here we review their structure, synthesis, photochemistry, photoproduct coordination chemistry and explore the potential biological and ecological consequences of this photoactivity.

Loss of Motility as a Non-Lethal Mechanism for Intercolony Inhibition ("Sibling Rivalry") in Marinobacter.

Bacteria from the genus Marinobacter are ubiquitous throughout the worlds' oceans as "opportunitrophs" capable of surviving a wide range of conditions, including colonization of surfaces of marine snow and algae. To prevent too many bacteria from occupying this ecological niche simultaneously, some sort of population dependent control must be operative. Here, we show that while Marinobacter do not produce or utilize an acylhomoserine lactone (AHL)-based quorum sensing system, "sibling" colonies of many species of Marinobacter exhibit a form of non-lethal chemical communication that prevents colonies from overrunning each other's niche space. Evidence suggests that this inhibition is the result of a loss in motility for cells at the colony interfaces. Although not the signal itself, we have identified a protein, glycerophosphoryl diester phosphodiesterase, that is enriched in the inhibition zone between the spreading colonies that may be part of the overall response.

Vibrioferrin, an unusual marine siderophore: iron binding, photochemistry, and biological implications.

Vibrioferrin (VF) is a member of the carboxylate class of siderophores originally isolated from Vibrio parahaemolyticus, an enteropathogenic estuarine bacterium often associated with seafood-borne gastroenteritis. Recently we have also isolated this siderophore from several species of Marinobacter, which are closely associated or "symbiotic" with toxic, bloom-forming dinoflagellates such as Gymnodinium catenatum. We have measured the overall metal-ligand binding constant for iron-vibrioferrin (FeVF) as 10(24.02(5)) making vibrioferrin one of the weakest iron chelators of any known marine siderophore. FeVF is also shown to be considerably more sensitive to photolysis under relatively low illumination conditions than other photoactive siderophores leading primarily to a monodecarboxylated photoproduct that has no significant affinity for Fe(III). The consequences that these features have on bacterial-algal interactions with potential importance to understanding the origin and sustenance of harmful algal blooms are discussed.

Ferric stability constants of representative marine siderophores: marinobactins, aquachelins, and petrobactin.

The coordination of iron(III) to the marine amphiphilic marinobactin and aquachelin siderophores, as well as to petrobactin, an unusual 3,4-dihydroxybenzoyl siderophore is reported. Potentiometric titrations were performed on the apo siderophore to determine the ligand pK(a) values, as well as the complex formed with addition of 1 equiv of Fe(III). The log K(ML) values for Fe(III)-marinobactin-E and Fe(III)-aquachelin-C are 31.80 and 31.4, respectively, consistent with the similar coordination environment in each complex, while log K(ML) for Fe(III)-petrobactin is estimated to be about 43. The pK(a) of the beta-hydroxyaspartyl hydroxyl group was determined to be 10.8 by (1)H NMR titration. (13)C NMR and IR spectroscopy were used to investigate Ga(III) coordination to the marinobactins. The coordination-induced shifts (CIS) in the (13)C NMR spectrum of Ga(III)-marinobactin-C compared to apo-marinobactin-C indicates that the hydroxamate groups are coordinated to Ga(III); however, the lack of CISs for the carbons of the beta-hydroxyamide group suggests this moiety is not coordinated in the Ga(III) complex. Differences in the IR spectrum of Ga(III)-marinobactin-C and Fe(III)-marinobactin-C in the 1600-1700 cm(-1) region also corroborates Fe(III) is coordinated to the beta-hydroxyamide moiety, whereas Ga(III) is not coordinated.