Genomic mosaicism underlies the adaptation of marine Synechococcus ecotypes to distinct oceanic iron niches

Phytoplankton are limited by iron (Fe) in ~40% of the world's oceans including high-nutrient low-chlorophyll (HNLC) regions. While low-Fe adaptation has been well-studied in large eukaryotic diatoms, less is known for small, prokaryotic marine picocyanobacteria. This study reveals key physiological and genomic differences underlying Fe adaptation in marine picocyanobacteria. HNLC ecotype CRD1 strains have greater physiological tolerance to low Fe congruent with their expanded repertoire of Fe transporter, storage and regulatory genes compared to other ecotypes. From metagenomic analysis, genes encoding ferritin, flavodoxin, Fe transporters and siderophore uptake genes were more abundant in low-Fe waters, mirroring paradigms of low-Fe adaptation in diatoms. Distinct Fe-related gene repertories of HNLC ecotypes CRD1 and CRD2 also highlight how coexisting ecotypes have evolved independent approaches to life in low-Fe habitats. Synechococcus and Prochlorococcus HNLC ecotypes likewise exhibit independent, genome-wide reductions of predicted Fe-requiring genes. HNLC ecotype CRD1 interestingly was most similar to coastal ecotype I in Fe physiology and Fe-related gene content, suggesting populations from these different biomes experience similar Fe-selective conditions. This work supports an improved perspective that phytoplankton are shaped by more nuanced Fe niches in the oceans than previously implied from mostly binary comparisons of low- versus high-Fe habitats and populations.     

铁施肥促进海洋浮游植物和生物碳泵的不确定性

铁作为浮游植物所必需的微量元素,限制了全球超过三分之一海域的初级生产力,尤其是在高营养盐、低叶绿素海域(high nutrient low chlorophyll,HNLC)。长期以来海洋铁施肥被认为是一项可以降低大气二氧化碳含量的地球工程策略。然而通过13次海洋人工铁施肥(artificial ocean iron fertilization,aOIF)实验发现,铁的额外添加对海洋深层碳输出量的促进作用要显著低于预期。本文简要地总结了碳在海洋和大气中的循环过程,回顾了人工铁施肥实验对生物碳泵和碳通量等的影响,分析了从海洋铁施肥到海洋碳汇关键生物地球化学过程的影响因素。综上分析发现,科学界对生物碳泵过程及其调控机制的认识仍十分浅薄,考虑到海洋铁施肥还会对海洋生态系统带来一定的负面作用,铁施肥能否作为降低大气中CO₂的有效手段,以达到碳中和并缓解温室效应仍需进一步研究。     

Ocean iron fertilization may amplify climate change pressures on marine animal biomass for limited climate benefit

Climate change scenarios suggest that large-scale carbon dioxide removal (CDR) will be required to maintain global warming below 2°C, leading to renewed attention on ocean iron fertilization (OIF). Previous OIF modelling has found that while carbon export increases, nutrient transport to lower latitude ecosystems declines, resulting in a modest impact on atmospheric CO₂. However, the interaction of these CDR responses with ongoing climate change is unknown. Here, we combine global ocean biogeochemistry and ecosystem models to show that, while stimulating carbon sequestration, OIF may amplify climate-induced declines in tropical ocean productivity and ecosystem biomass under a high-emission scenario, with very limited potential atmospheric CO₂ drawdown. The ‘biogeochemical fingerprint’ of climate change, that leads to depletion of upper ocean major nutrients due to upper ocean stratification, is reinforced by OIF due to greater major nutrient consumption. Our simulations show that reductions in upper trophic level animal biomass in tropical regions due to climate change would be exacerbated by OIF within ~20 years, especially in coastal exclusive economic zones (EEZs), with potential implications for fisheries that underpin the livelihoods and economies of coastal communities. Any fertilization-based CDR should therefore consider its interaction with ongoing climate-driven changes and the ensuing ecosystem impacts in national EEZs.     

Iron-Stimulated N(2) Fixation and Growth in Natural and Cultured Populations of the Planktonic Marine Cyanobacteria Trichodesmium spp

In light of recent proposals that iron (Fe) availability may play an important role in controlling oceanic primary production and nutrient flux, its regulatory impact on N(2) fixation and production dynamics was investigated in the widespread and biogeochemically important diazotrophic, planktonic cyanobacteria Trichodesmium spp. Fe additions, as FeCl(3) and EDTA-chelated FeCl(3), enhanced N(2) fixation (nitrogenase activity), photosynthesis (CO(2) fixation), and growth (chlorophyll a production) in both naturally occurring and cultured (on unenriched oligotrophic seawater) Trichodesmium populations. Maximum enhancement of these processes occurred under FeEDTA-amended conditions. On occasions, EDTA alone led to enhancement. No evidence for previously proposed molybdenum or phosphorus limitation was found. Our findings geographically extend support for Fe limitation of N(2) fixation and primary production to tropical and subtropical oligotrophic ocean waters often characterized by Trichodesmium blooms.     

Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet, British Columbia

Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the δ- proteobacteria , SAR406 and γ- proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.     

A novel haloarchaeal-related lineage is widely distributed in deep oceanic regions

During our study of the 16S rRNA gene sequence-based archaeal diversity of a deep-sea site located at a 3000 m depth at the Antarctic Polar Front, we detected several phylotypes ascribed to already known Group II and III Euryarchaeota, and a cluster of distinct sequences that branched off at the base of haloarchaea. The position of this lineage (marine Group IV) was very robust using distance (neighbour-joining) and maximum-likelihood methods. Subsequently, we designed specific primers for the detection of this archaeal group in other marine environments using polymerase chain reaction amplification and sequence comparison. Group IV archaea were found in the Antarctic area (across a gradient from the Southern ocean to the South Atlantic), and also in North Atlantic and Mediterranean waters. In all oceanic locations, Group IV archaea were never detected in surface waters, but were vertically distributed in the deepest part of the water column.     

Major differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean

One of the first comparisons of a natural iron fertilized bloom with a high-nutrient low-chlorophyll (HNLC) site was undertaken during the Kerguelen ocean and plateau compared study (KEOPS) cruise. To understand better the bacteria–phytoplankton relationship in the context of natural iron fertilization, bacterial diversity and activity was investigated in the bloom and in the adjacent HNLC region by 16S rDNA clone libraries and by single strand conformation polymorphism (SSCP) analysis. Both libraries were dominated by Alphaproteobacteria , Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cluster analysis at 99% sequence similarity yielded several microdiverse clusters and revealed striking differences between the two libraries. In the bloom, the dominant operational taxonomic units (OTUs) were the Roseobacter NAC11-7 cluster, SAR92 and a Cytophaga-Flavobacteria-Bacteroides cluster related to the agg58 group, whereas in the HNLC region, SAR11, Roseobacter RCA and Polaribacter dominated. SSCP analysis of 16S rDNA and 16S rRNA revealed contrasting dynamics of three different Roseobacter OTUs. Roseobacter NAC11-7 and NAC11-6 had higher relative abundances and activities in the bloom compared with the HNLC site and NAC11-6 was only detected at the decline of the bloom concomitant with a shift in phytoplankton composi tion. In contrast, Roseobacter RCA was relatively abundant and active both inside and outside of the bloom. These results suggest that the different OTUs within the Roseobacter group represent functional groups that each play an important role in the cycling of carbon.     

Lake Biwa and the ocean: geochemical similarity and difference

The average composition of water, bottom sediments, manganese (Mn) crusts, and Mn concretions from Lake Biwa (the largest freshwater lake in Japan) are re-examined, in conjunction with those of seawater, oceanic pelagic clay, and deep-sea Mn nodules. The purpose is to gain additional insights into the geochemical behaviors of elements in Lake Biwa and the ocean, which are quite different in ionic strength (or salinity), pH, water residence times, sediment accumulation rates, carbon fluxes to sediments, and the redox potential in sediments. Excluding a few millimeters of oxic surface sediment, there is no appreciable accumulation of Mn in the Lake Biwa bottom sediments due to reducing condition there. Consequently, other B-type cations [such as iron (Fe), gallium (Ga), copper (Cu), lead (Pb), cobalt (Co), tin (Sn), and bismuth (Bi), with subshell valence electron configuration of d ¹⁻¹⁰] are also less concentrated in the lake sediments than in the oceanic pelagic clay. In turn, B-type cations have much higher dissolved concentrations in the lake water than in the ocean. The rare earth elements (REE) mainly form organic complexes in the lake water and carbonate complexes in the ocean. REE are mostly associated with detritus aluminosilicate phases in Lake Biwa sediments but with phosphate phases in deep-sea sediments. Fe and Mn oxide phases are clearly separated in marine Mn nodules and crusts but not in Mn crusts and concretions from Lake Biwa. Useful parameters such as the enrichment factor (E _Al) and logarithms of the distribution coefficient (log K _d) of elements between solid and liquid phases were estimated in both systems for further discussions.     

Iron-Stimulated N2 Fixation and Growth in Natural and Cultured Populations of the Planktonic Marine Cyanobacteria Trichodesmium spp

In light of recent proposals that iron (Fe) availability may play an important role in controlling oceanic primary production and nutrient flux, its regulatory impact on N₂ fixation and production dynamics was investigated in the widespread and biogeochemically important diazotrophic, planktonic cyanobacteria Trichodesmium spp. Fe additions, as FeCl₃ and EDTA-chelated FeCl₃, enhanced N₂ fixation (nitrogenase activity), photosynthesis (CO₂ fixation), and growth (chlorophyll a production) in both naturally occurring and cultured (on unenriched oligotrophic seawater) Trichodesmium populations. Maximum enhancement of these processes occurred under FeEDTA-amended conditions. On occasions, EDTA alone led to enhancement. No evidence for previously proposed molybdenum or phosphorus limitation was found. Our findings geographically extend support for Fe limitation of N₂ fixation and primary production to tropical and subtropical oligotrophic ocean waters often characterized by Trichodesmium blooms.     

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.     

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.     

Individual foraging site fidelity in lactating New Zealand fur seals: Continental shelf vs. oceanic habitats

Wide‐ranging marine central place foragers often exhibit foraging site fidelity to oceanographic features over differing spatial scales (i.e., localized coastal upwellings and oceanic fronts). Few studies have tested how the degree of site fidelity to foraging areas varies in relation to the type of ocean features used. In order to determine how foraging site fidelity varied between continental shelf and oceanic foraging habitats, 31 lactating New Zealand fur seals (Arctocephalus australis forsteri 1 ) were satellite tracked over consecutive foraging trips (14–108 d). Thirty‐seven foraging trips were recorded from 11 females that foraged on the continental shelf, in a region associated with a coastal upwelling, while 65 foraging trips were recorded from 20 females that foraged in oceanic waters. There were no significant differences in the mean bearings (to maximum distance) of individual's consecutive foraging trips, suggesting individual fidelity to foraging areas. However, overlap in area and time spent in area varied considerably between continental shelf and oceanic foragers. Females that foraged on the continental shelf had significantly greater overlap in consecutive foraging trips when compared to females that foraged in oceanic waters (overlap in 5 × 5 km grid cells visited on consecutive trips 55.9%± 20.4% and 13.4%± 7.6%, respectively). Females that foraged on the continental shelf also spent significantly more time within the same grid cell than females that foraged in oceanic waters (maximum time spent in 5 × 5 km grid cells: 14%± 5% and 4%± 2%, respectively). This comparatively high foraging site fidelity may reflect the concentration of productivity associated with a coastal upwelling system, the Bonney Upwelling. Lower foraging site fidelity recorded by seals that foraged in oceanic waters implies a lower density/larger scale habitat, where prey are more dispersed or less predictable at fine scales, when compared to the continental shelf region.     

Mechanism of Ferrous Iron Binding and Oxidation by Ferritin from a Pennate Diatom

A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.     

Variation in nutrient limitation and seagrass nutrient content in Bahamian tidal creek ecosystems

The traditional model of nutrient availability in coastal estuarine ecosystems is based on predictable inputs of nitrogen (N) and phosphorus (P) via riverine and oceanic sources, respectively. But coastlines with low nutrient input from these sources may not fit into this simple framework. Here we use observational (seagrass nutrient content) and experimental (nutrient enrichment assays) data for assessing nutrient availability and limitation for primary producers along a spatial transect extending from the mouth (nearest to the ocean) to the terminal portion (boundary with the terrestrial ecosystem) of three coastal mangrove-lined tidal creeks in The Bahamas. Compiling seagrass nutrient content from all sites showed a negative relationship between seagrass nutrient limitation (either N or P) and distance from mouth, but this pattern differed across sites with respect to which nutrient was more limiting. Our experimental results demonstrated patterns of decreased response by microalgae to dual nutrient enrichment in one site with distance from the creek mouth, and increased response to single nutrient enrichment in another, with the third showing no trend along this gradient. Our findings show that Bahamian mangrove wetlands are extremely nutrient-limited ecosystems, and that the most limiting nutrient varied among sites. In general, these ecosystems deviate from the typical paradigm of spatial nutrient limitation patterns in estuaries. We suggest that various site-specific biological and physical factors may be more important than large-scale hydrologic factors in driving trends of nutrient availability in coastal ecosystems under strong nutrient constraints, such as in The Bahamas. Our findings suggest that even minor changes in nutrient loading rates can have significant implications for primary production in subtropical oligotrophic systems.     

Exponential decline of deep-sea ecosystem functioning linked to benthic biodiversity loss

BACKGROUND: Recent investigations suggest that biodiversity loss might impair the functioning and sustainability of ecosystems. Although deep-sea ecosystems are the most extensive on Earth, represent the largest reservoir of biomass, and host a large proportion of undiscovered biodiversity, the data needed to evaluate the consequences of biodiversity loss on the ocean floor are completely lacking. RESULTS: Here, we present a global-scale study based on 116 deep-sea sites that relates benthic biodiversity to several independent indicators of ecosystem functioning and efficiency. We show that deep-sea ecosystem functioning is exponentially related to deep-sea biodiversity and that ecosystem efficiency is also exponentially linked to functional biodiversity. These results suggest that a higher biodiversity supports higher rates of ecosystem processes and an increased efficiency with which these processes are performed. The exponential relationships presented here, being consistent across a wide range of deep-sea ecosystems, suggest that mutually positive functional interactions (ecological facilitation) can be common in the largest biome of our biosphere. CONCLUSIONS: Our results suggest that a biodiversity loss in deep-sea ecosystems might be associated with exponential reductions of their functions. Because the deep sea plays a key role in ecological and biogeochemical processes at a global scale, this study provides scientific evidence that the conservation of deep-sea biodiversity is a priority for a sustainable functioning of the worlds' oceans.     

Analysis of the global ocean sampling (GOS) project for trends in iron uptake by surface ocean microbes

Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe(2+) versus Fe(3+) uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.     

Contribution of picoplankton to primary production in estuarine,coastal and equatorial waters of Brazil

Quantitative data on primary production of picoplankton are presented for three different Brazilian marine ecosystems: estuarine, coastal and oceanic. The size fraction from 0.45 μm to 1.0 μm was responsible for 3.0% to 28.5% of ¹⁴C uptake in estuarine waters; 18.5% to 40.4% in coastal waters, and 6.7% to 100.0% in oceanic waters, respectively. These results establish the importance of picoplankton as primary producers in different marine environments along the Brazilian waters.     

Altered Sea Ice Thickness and Permanence Affects Benthic Ecosystem Functioning in Coastal Antarctica

Antarctic sea ice and the cold waters surrounding the continent are key elements of the global climate system, influencing heat redistribution, oceanic circulation and the absorption of carbon dioxide from the atmosphere. However, the Southern Ocean is predicted to warm by 1–6°C over the next century, altering sea ice extent, thickness and permanence. To better understand the connections between coastal sea ice conditions and the functioning of Antarctica’s unique marine benthic ecosystems, we performed manipulative experiments on the seafloor at two southwestern Ross Sea sites with contrasting sea ice conditions. Benthic systems at both study sites were net heterotrophic during the study period (early November), with primary production most likely limited by light availability rather than nutrients. There was five times more fresh algal detrital material in benthic sediments at the site with the thinner, snow-free, annually formed sea ice, relative to the site with thicker, multiyear sea ice. This elevated quantity and quality of algal detrital matter corresponded with a significantly greater rate of sediment oxygen utilization by the benthos and an altered pathway of nitrogen regeneration (tighter coupling between nitrification and denitrification). Large benthic animals (brittle stars, Ophionotus victoriae) enhanced the efflux of dissolved inorganic nutrients from the sediment to the water column and played a greater role in nutrient regeneration at the site with more food. Although changes in sea ice characteristics in the Western Ross Sea are difficult to predict at present, large benthic organisms can be expected to have an expanded role in mediating the effects of elevated coastal productivity and detritus supply on ecosystem dynamics in this part of Antarctica.     

Decreased precipitation exacerbates the effects of sea level on coastal dune ecosystems in open ocean islands

The alteration of fresh and marine water cycling is likely to occur in coastal ecosystems as climate change causes the global redistribution of precipitation while simultaneously driving sea‐level rise at a rate of 2–3 mm yr^?1. Here, we examined how precipitation alters the ecological effects of ocean water intrusion to coastal dunes on two oceanic carbonate islands in the Bahamas. The approach was to compare sites that receive high and low annual rainfall and are also characterized by seasonal distribution (wet and dry season) of precipitation. The spatial and temporal variations in precipitation serve as a proxy for conditions of altered precipitation which may occur via climate change. We used the natural abundances of stable isotopes to identify water sources (e.g., precipitation, groundwater and ocean water) in the soil–plant continuum and modeled the depth of plant water uptake. Results indicated that decreased rainfall caused the shallow freshwater table on the dune ecosystem to sink and contract towards the inland, the lower freshwater head allowed ocean water to penetrate into the deeper soils, while shallow soils became exceedingly dry. Plants at the drier site that lived nearest to the ocean responded by taking up water from the deeper and consistently moist soil layers where ocean water intruded. Towards the inland, decreased rainfall caused the water table to sink to a depth that precluded both recharge to the upper soil layers and access by plants. Consequently, plants captured water in more shallow soils recharged by infrequent rainfall events. The results demonstrate dune ecosystems on oceanic islands are more susceptible to ocean water intrusion when annual precipitation decreases. Periods of diminished precipitation caused drought conditions, increased exposure to saline marine water and altered water‐harvesting strategies. Quantifying species tolerances to ocean water intrusion and drought are necessary to determine a threshold of community sustainability.     

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)