John R. Reinfelder

The evolutionary inheritance of elemental stoichiometry in marine phytoplankton

Phytoplankton is a nineteenth century ecological construct for a biologically diverse group of pelagic photoautotrophs that share common metabolic functions but not evolutionary histories. In contrast to terrestrial plants, a major schism occurred in the evolution of the eukaryotic phytoplankton that gave rise to two major plastid superfamilies. The green superfamily appropriated chlorophyll b, whereas the red superfamily uses chlorophyll c as an accessory photosynthetic pigment. Fossil evidence suggests that the green superfamily dominated Palaeozoic oceans. However, after the end-Permian extinction, members of the red superfamily rose to ecological prominence. The processes responsible for this shift are obscure. Here we present an analysis of major nutrients and trace elements in 15 species of marine phytoplankton from the two superfamilies. Our results indicate that there are systematic phylogenetic differences in the two plastid types where macronutrient (carbon:nitrogen:phosphorus) stoichiometries primarily reflect ancestral pre-symbiotic host cell phenotypes, but trace element composition reflects differences in the acquired plastids. The compositional differences between the two plastid superfamilies suggest that changes in ocean redox state strongly influenced the evolution and selection of eukaryotic phytoplankton since the Proterozoic era.

Bioaccumulation of mercury and methylmercury

The factors controlling the accumulation of mercury in fish are poorly understood. The oil invoked lipid solubility of MMHg is an inadequate explanation because inorganic Hg complexes, which are not bioaccumulated, are as lipid soluble as their MMHg analogs and, unlike other hydrophobic compounds, MMHg in fish resides in protein rather than fat tissue. We show that passive uptake of the lipophilic complexes (primarily HgCl2 and CH3HgCl) results in high concentrations of both inorganic and MMHg in phytoplankton. However, differences in partitioning within phytoplankton cells between inorganic mercury — which is principally membrane bound — and MMHg — which accumulates in the cytoplasm — lead to a greater assimilation of MMHg during zooplankton grazing. Most of the discrimination between inorganic and MMHg thus occurs during trophic transfer while the major enrichment factor is between water and phytoplankton. As a result, MMHg concentrations in fish are ultimately determined by water chemistry which controls MMHg speciation and uptake at the base of the food chain.

The Assimilation of Elements Ingested by Marine Copepods

The efficiency with which a variety of ingested elements (Ag, Am, C, Cd, P, S, Se, and Zn) were assimilated in marine calanoid copepods fed uniformly radiolabeled diatoms ranged from 0.9% for Am to 97.1% for Se. Assimilation efficiencies were directly related to the cytoplasmic content of the diatoms. This relation indicates that the animals obtained nearly all their nutrition from this source. The results suggest that these zooplankton, which have short gut residence times, have developed a gut lining and digestive strategy that provides for assimilation of only soluble material. Because the fraction of total cellular protein in the cytoplasm of the diatoms increased markedly with culture age, copepods feeding on senescent cells should obtain more protein than those feeding on rapidly dividing cells. Elements that are appreciably incorporated into algal cytoplasm and assimilated in zooplankton should be recycled in surface waters and have longer oceanic residence times than elements bound to cell surfaces.

Trace element trophic transfer in aquatic organisms: A critique of the kinetic model approach

The bioaccumulation of trace elements in aquatic organisms can be described with a kinetic model that includes linear expressions for uptake and elimination from dissolved and dietary sources. Within this model, trace element trophic transfer is described by four parameters: the weight-specific ingestion rate (IR); the assimilation efficiency (AE); the physiological loss rate constant (ke); and the weight-specific growth rate (g). These four parameters define the trace element trophic transfer potential (TTP = IR.AE/[ke + g]) which is equal to the ratio of the steady-state trace element concentration in a consumer due to trophic accumulation to that in its prey. Recent work devoted to the quantification of AE and ke for a variety of trace elements in aquatic invertebrates has provided the data needed for comparative studies of trace element trophic transfer among different species and trophic levels and, in at least one group of aquatic consumers (marine bivalves), sensitivity analyses and field tests of kinetic bioaccumulation models. Analysis of the trophic transfer potentials of trace elements for which data are available in zooplankton, bivalves, and fish, suggests that slight variations in assimilation efficiency or elimination rate constant may determine whether or not some trace elements (Cd, Se, and Zn) are biomagnified. A linear, single-compartment model may not be appropriate for fish which, unlike many aquatic invertebrates, have a large mass of tissue in which the concentrations of most trace elements are subject to feedback regulation.

Unicellular C4 photosynthesis in a marine diatom.

Nearly 50 years ago, inorganic carbon was shown to be fixed in microalgae as the C3 compound phosphoglyceric acid. The enzyme responsible for C3 carbon fixation, ribulose-1,5-bisphosphate carboxylase (Rubisco), however, requires inorganic carbon in the form of CO2 (ref. 2), and Rubisco enzymes from diatoms have half-saturation constants for CO2 of 30–60 µM (ref. 3). As a result, diatoms growing in seawater that contains about 10 µM CO2 may be CO2 limited. Kinetic and growth studies have shown that diatoms can avoid CO2 limitation, but the biochemistry of the underlying mechanisms remains unknown. Here we present evidence that C4 photosynthesis supports carbon assimilation in the marine diatom Thalassiosira weissflogii, thus providing a biochemical explanation for CO2-insensitive photosynthesis in marine diatoms. If C4 photosynthesis is common among marine diatoms, it may account for a significant portion of carbon fixation and export in the ocean, and would explain the greater enrichment of 13C in diatoms compared with other classes of phytoplankton. Unicellular C4 carbon assimilation may have predated the appearance of multicellular C4 plants.

Active uptake of bicarbonate by diatoms

Marine diatoms play a predominant role in the biological carbon pump transferring carbon dioxide from surface to deep waters. Laboratory studies show that a number of species take up HCO3− and concentrate inorganic carbon intracellularly allowing rapid growth despite low CO2 availability,. In contrast, many oceanographers, particularly when interpreting carbon isotope data,, have made the assumption that diatoms do not utilize the abundant HCO3− in seawater but rather take up CO2 by diffusion. This has led to the hypothesis that large diatoms may be CO2-limited in the oceans. We now demonstrate active uptake of HCO3− in the field and a carbon-concentrating mechanism in coastal Atlantic diatoms. By manipulating pCO2 we show that growth of large diatoms in the California upwelling is not limited by CO2 availability.

The Role of the C4 Pathway in Carbon Accumulation and Fixation in a Marine Diatom

The role of a C4 pathway in photosynthetic carbon fixation by marine diatoms is presently debated. Previous labeling studies have shown the transfer of photosynthetically fixed carbon through a C4 pathway and recent genomic data provide evidence for the existence of key enzymes involved in C4 metabolism. Nonetheless, the importance of the C4 pathway in photosynthesis has been questioned and this pathway is seen as redundant to the known CO2 concentrating mechanism of diatoms. Here we show that the inhibition of phosphoenolpyruvate carboxylase (PEPCase) by 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate resulted in a more than 90% decrease in whole cell photosynthesis in Thalassiosira weissflogii cells acclimated to low CO2 (10 μm), but had little effect on photosynthesis in the C3 marine Chlorophyte, Chlamydomonas sp. In 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate-treated T. weissflogii cells, elevated CO2 (150 μm) or low O2 (80–180 μm) restored photosynthesis to the control rate linking PEPCase inhibition with CO2 supply in this diatom. In C4 organic carbon-inorganic carbon competition experiments, the 12C-labeled C4 products of PEPCase, oxaloacetic acid and its reduced form malic acid suppressed the fixation of 14C-labeled inorganic carbon by 40% to 50%, but had no effect on O2 evolution in photosynthesizing diatoms. Oxaloacetic acid-dependent O2 evolution in T. weissflogii was twice as high in cells acclimated to 10 μm rather than 22 μm CO2, indicating that the use of C4 compounds for photosynthesis is regulated over the range of CO2 concentrations observed in marine surface waters. Short-term 14C uptake (silicone oil centrifugation) and CO2 release (membrane inlet mass spectrometry) experiments that employed a protein denaturing cell extraction solution containing the PEPCKase inhibitor mercaptopicolinic acid revealed that much of the carbon taken up by diatoms during photosynthesis is stored as organic carbon before being fixed in the Calvin cycle, as expected if the C4 pathway functions as a CO2 concentrating mechanism. Together these results demonstrate that the C4 pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low (atmospheric) CO2.

Acquisition of inorganic carbon by the marine diatom Thalassiosira weissflogii

Recent data on the physiology of inorganic carbon acquisition by the model marine diatom Thalassiosira weissflogii (Grunow) demonstrate the importance of the catalytic equilibration of HCO3-and CO2by carbonic anhydrases located in the periplasm and in the cytoplasm. These enzymes can use Zn, Co or Cd as their metal centre, and their activity increases at low ambient CO2. The silica frustule provides buffering for extracellular CA activity, The transmembrane transport of CO2 may occur by passive diffusion. Under CO2 limitation, the cytoplasmic HCO3-is used to form malate and oxaloacetic acid via phosphoenolpyruvate carboxylase. It appears that subsequent decarboxylation of these compounds in the chloroplast regenerates CO2 near the site of Rubisco, and thus provides the organism with an effective unicellular C4 photosynthetic pathway. These results, together with other published data, bring up two major questions regarding inorganic carbon acquisition in diatoms: What is the major species of inorganic carbon (CO2 or HCO3-) transported across the membrane under natural conditions? And what is the form of carbon (inorganic or organic) accumulated by the cells?

Localization and Role of Manganese Superoxide Dismutase in a Marine Diatom

Superoxide dismutase (SOD) catalyzes the transformation of superoxide to molecular oxygen and hydrogen peroxide. Of the four known SOD isoforms, distinguished by their metal cofactor (iron, manganese [Mn], copper/zinc, nickel), MnSOD is the dominant form in the diatom Thalassiosira pseudonana. We cloned the MnSOD gene, sodA, using the expression vector pBAD, overexpressed the product in Escherichia coli, and purified the mature protein (TpMnSOD). This recombinant enzyme was used to generate a polyclonal antibody in rabbit that recognizes MnSOD in T. pseudonana. Based on quantitative immunoblots, we calculate that in vivo concentrations of TpMnSOD are approximately 0.9 amol cell−1 using the recombinant protein as a standard. Immunogold staining indicates that TpMnSOD is localized in the chloroplasts, which is in contrast to most other eukaryotic algae (including chlorophytes and embryophytes) where MnSOD is localized exclusively in mitochondria. Based on the photosynthetic Mn complex in photosystem II, cellular Mn budgets cannot account for 50% to 80% of measured Mn within diatom cells. Our results reveal that chloroplastic MnSOD accounts for 10% to 20% of cellular Mn, depending on incident light intensity and cellular growth rate. Indeed, our analysis indicates that TpMnSOD accounts for 1.4% (±0.2%) of the total protein in the cell. The TpMnSOD has a rapid turnover rate with an apparent half-life of 6 to 8 h when grown under continuous light. TpMnSOD concentrations increase relative to chlorophyll, with an increase in incident light intensity to minimize photosynthetic oxidative stress. The employment of a Mn-based SOD, linked to photosynthetic stress in T. pseudonana, may contribute to the continued success of diatoms in the low iron regions of the modern ocean.

Mercury Methylation by the Methanogen Methanospirillum hungatei

ABSTRACT Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.