Nitrogen and Iron Availability Drive Metabolic Remodeling and Natural Selection of Diverse Phytoplankton during Experimental Upwelling.

Nearly half of carbon fixation and primary production originates from marine phytoplankton, and much of it occurs in episodic blooms in upwelling regimes. Here, we simulated blooms limited by nitrogen and iron by incubating Monterey Bay surface waters with subnutricline waters and inorganic nutrients and measured the whole-community transcriptomic response during mid- and late-bloom conditions. Cell counts revealed that centric and pennate diatoms (largely Pseudo-nitzschia and Chaetoceros spp.) were the major blooming taxa, but dinoflagellates, prasinophytes, and prymnesiophytes also increased. Viral mRNA significantly increased in late bloom and likely played a role in the bloom's demise. We observed conserved shifts in the genetic similarity of phytoplankton populations to cultivated strains, indicating adaptive population-level changes in community composition. Additionally, the density of single nucleotide variants (SNVs) declined in late-bloom samples for most taxa, indicating a loss of intraspecific diversity as a result of competition and a selective sweep of adaptive alleles. We noted differences between mid- and late-bloom metabolism and differential regulation of light-harvesting complexes (LHCs) under nutrient stress. While most LHCs are diminished under nutrient stress, we showed that diverse taxa upregulated specialized, energy-dissipating LHCs in low iron. We also suggest the relative expression of NRT2 compared to the expression of GSII as a marker of cellular nitrogen status and the relative expression of iron starvation-induced protein genes (ISIP1, ISIP2, and ISIP3) compared to the expression of the thiamine biosynthesis gene (thiC) as a marker of iron status in natural diatom communities. IMPORTANCE Iron and nitrogen are the nutrients that most commonly limit phytoplankton growth in the world's oceans. The utilization of these resources by phytoplankton sets the biomass available to marine systems and is of particular interest in high-nutrient, low-chlorophyll (HNLC) coastal fisheries. Previous research has described the biogeography of phytoplankton in HNLC regions and the transcriptional responses of representative taxa to nutrient limitation. However, the differential transcriptional responses of whole phytoplankton communities to iron and nitrogen limitation has not been previously described, nor has the selective pressure that these competitive bloom environments exert on major players. In addition to describing changes in the physiology of diverse phytoplankton, we suggest practical indicators of cellular nitrogen and iron status for future monitoring.

Detection of a key Hg methylation gene, hgcA, in wetland soils.

The corrinoid protein, HgcA has been shown to be essential for Hg methylation in anaerobic bacteria. We investigated the diversity of hgcA from temperate and tropical wetland soils where Hg methylation is demonstrated. Sequences obtained from both environments clustered with those from the δ-Proteobacteria, Chloroflexi and Methanomicrobia with significant overlap in hgcA phylogeny between libraries. Clear differences in hgcA distribution were observed between two highly contrasting sites within a tropical wetland in Everglades National Park, USA. hgcA sequences obtained from the northern site clustered primarily with those of methanogens, while sequences from the estuarine site clustered primarily with sulphate-reducing bacteria and syntrophs in the δ-Proteobacteria. Libraries obtained from soils collected from a temperate swamp in Sweden were dominated by hgcA sequences within the δ-Proteobacteria with hgcA sequences clustering primarily with iron reducers in the upstream portion of the swamp and with sulphate reducers in the downstream portion of the swamp. Interestingly, enrichments prepared from the lower portion of this temperate wetland contained a high abundance of hgcA sequences clustering with methanogens. This first report on hgcA diversity in environmental samples suggests a role in Hg methylation for various phenotypic groups in different portions of wetlands.

Multiple roles of siderophores in free-living nitrogen-fixing bacteria.

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.

Overexpression and characterization of an iron storage and DNA-binding Dps protein from Trichodesmium erythraeum.

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N(2)-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps(T. erythraeurm) protein (Dps(tery)) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps(tery), and we found that Dps(tery), like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps(tery) binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps(tery) indicated that it has an iron core that resembles that of horse spleen ferritin.

The biogeochemical cycles of trace metals in the oceans.

Planktonic uptake of some essential metals results in extraordinarily low concentrations in surface seawater. To sequester or take up these micronutrients, various microorganisms apparently release strong complexing agents and catalyze redox reactions that modify the bioavailability of trace metals and promote their rapid cycling in the upper water column. In turn, the low availability of some metals controls the rate of photosynthesis in parts of the oceans and the transformation and uptake of major nutrients such as nitrogen. The extremely low concentrations of several essential metals are both the cause and the result of ultraefficient uptake systems in the plankton and of widespread replacement of metals by one another for various biochemical functions.

Phytochelatins are bioindicators of atmospheric metal exposure via direct foliar uptake in trees near Sudbury, Ontario, Canada.

Plants produce phytochelatins in response to copper and nickel, the primary metal pollutants emitted by the dominant smelting operation in Sudbury. Copper and nickel concentrations in soils decline sharply with distance from this facility, primarily as a result of early smelting practices. Phytochelatin concentrations in Sudbury-area trees, however, do not correlate with metal levels in soils. Rather, phytochelatin production in tree leaves is driven by metals currently released to the atmosphere through the 381 m emissions stack. Phytochelatin concentrations in the foliage of three tree species growing in situ are highest 20-30 km from the stack, correlated with maximum acid-leachable concentrations of deposited copper and nickel. Similar results observed in potted trees placed adjacent to indigenous trees confirm that aerially deposited metals are the source of current metal stress patterns. The addition of peat moss "filters" to potted soils did not alter this response, indicating that direct foliar metal uptake is responsible.

Photooxidation of Hg(0) in artificial and natural waters.

The oxidation of volatile aqueous Hg(0) in aquatic systems may be important in reducing fluxes of Hg out of aquatic systems. Here we report the results of laboratory and field experiments designed to identify the parameters that control the photooxidation of Hg(0)(aq) and to assess the possible importance of this process in aquatic systems. The concentrations of elemental and total Hg were measured as a function of time in both artificial and natural waters irradiated with a UV-B lamp. No change in Hg speciation was observed in dark controls, while a significant decrease in Hg(0) was observed in UV-B irradiated artificial solutions containing both chloride ions and benzoquinone. Significant photooxidation rates were also measured in natural samples spiked with Hg(0)(aq); the photooxidation of Hg(0) then follows pseudo first-order kinetics (k = 0.6 h(-1)). These results indicate that the previously observed Hg(II) photoreduction rates in natural waters could represent a net balance between Hg(0) photoreduction and Hg(0) photooxidation. As calculated from Hg(0) photooxidation rates, the dominant Hg(0) sink is likely to be photooxidation rather than volatilization from the water column during summer days.

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 microM (ref. 3). As a result, diatoms growing in seawater that contains about 10 microM 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.

Reduced calcification of marine plankton in response to increased atmospheric CO2.

The formation of calcareous skeletons by marine planktonic organisms and their subsequent sinking to depth generates a continuous rain of calcium carbonate to the deep ocean and underlying sediments. This is important in regulating marine carbon cycling and ocean-atmosphere CO2 exchange. The present rise in atmospheric CO2 levels causes significant changes in surface ocean pH and carbonate chemistry. Such changes have been shown to slow down calcification in corals and coralline macroalgae, but the majority of marine calcification occurs in planktonic organisms. Here we report reduced calcite production at increased CO2 concentrations in monospecific cultures of two dominant marine calcifying phytoplankton species, the coccolithophorids Emiliania huxleyi and Gephyrocapsa oceanica. This was accompanied by an increased proportion of malformed coccoliths and incomplete coccospheres. Diminished calcification led to a reduction in the ratio of calcite precipitation to organic matter production. Similar results were obtained in incubations of natural plankton assemblages from the north Pacific ocean when exposed to experimentally elevated CO2 levels. We suggest that the progressive increase in atmospheric CO2 concentrations may therefore slow down the production of calcium carbonate in the surface ocean. As the process of calcification releases CO2 to the atmosphere, the response observed here could potentially act as a negative feedback on atmospheric CO2 levels.

The active site structure of Thalassiosira weissflogii carbonic anhydrase 1.

X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii.

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.

A biological function for cadmium in marine diatoms.

The oceanic distribution of cadmium follows closely that of major algal nutrients such as phosphate. The reasons for this "nutrient-like" distribution are unclear, however, because cadmium is not generally believed to have a biological function. Herein, we provide evidence of a biological role for Cd in the marine diatom Thalassiosira weissflogii under conditions of low zinc, typical of the marine environment. Addition of Cd to Zn-limited cultures enhances the growth rate of T. weissflogii, particularly at low pCO(2). This increase in growth rate is reflected in increased levels of cellular carbonic anhydrase (CA) activity, although the levels of TWCA1, the major intracellular Zn-requiring isoform of CA in T. weissflogii, remain low. (109)Cd label comigrates with a protein band that shows CA activity and is distinct from TWCA1 on native PAGE of radiolabeled T. weissflogii cell lysates. The levels of the Cd protein are modulated by CO(2) in a manner that is consistent with a role for this enzyme in carbon acquisition. Purification of the CA-active fraction leads to the isolation of a Cd-containing protein of 43 kDa. It is now clear that T. weissflogii expresses a Cd-specific CA, which, particularly under conditions of Zn limitation, can replace the Zn enzyme TWCA1 in its carbon-concentrating mechanism.

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42. H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the epsilon-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.

Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay.

Phytochelatins are small metal-binding polypeptides synthesized by algae in response to high metal concentrations. Using a very sensitive HPLC method, we have quantified phytochelatins from phytoplankton in laboratory cultures at environmentally relevant metal concentrations and in marine field samples. Intracellular concentrations of phytochelatin, in the diatom Thalassiosira weissflogii, exhibit a distinct dose-response relation with free Cd2+ concentration in the medium--not with total Cd(2+)--and are detectable even when the free Cd2+ concentration is less than 1 pM. In Massachusetts Bay, phytochelatin levels (normalized to chlorophyll a) in the particulate fraction are similar to those measured in laboratory cultures exposed to picomolar free Cd2+ concentrations and exhibit a decreasing seaward trend. Incubations of natural samples with added Cd2+ confirmed the induction of the peptides by this metal. Ambient phytochelatin concentrations thus appear to provide a measure of the metal stress resulting from the complex mixture of trace metals and chelators in natural waters.

Amine oxidases of marine phytoplankton.

Some phytoplankton utilized a novel mechanism for obtaining nitrogen from primary amines. They oxidized the primary amines to produce extracellular hydrogen peroxide and aldehydes and used the third reaction product, ammonium, as a nitrogen source. The specificity, regulation, inhibition by bromoethylamine, and potential dependence on copper of this process are described.

Potential effects of UV-B on the chemical environment of marine organisms: a review.

An increase in ultraviolet-B (UV-B) due to depletion of stratospheric ozone may affect growth of marine phytoplankton by altering the chemistry of their environment. Production of bioactive free radicals, photodecomposition of organic matter, and availability of trace metals are likely to be altered by increased UV-B flux. Such changes to the chemical environment may be both deleterious and beneficial to marine phytoplankton. Extracellular free radicals such as OH, Br(2)(-), and CO(3)(-) are predicted to have a negligible impact, but superoxide and its decomposition product hydrogen peroxide may react rapidly with cell surfaces and destroy membrane function and integrity. Increased UV-B will enhance the bioavailability of the redox active trace metals Fe and Cu. Thus, in the Fe-limited high latitude ocean, increased Fe availability may promote phytoplankton production, while in other parts of the ocean increased Cu availability may be toxic. Overall, the interdependent direct and indirect effects of UV-B on phytoplankton may compensate for each other and account for the ability of marine ecosystems to be subjected to widely variable UV-B flux without apparent damage.

Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase.

Plasmalemma redox activity in the diatom Thalassiosira is competitively inhibited by antiserum prepared against algal nitrate reductase (NR), and fluorescent labeling experiments reveal the binding of NR antiserum to the cell surface. Furthermore, the external electron acceptor Cu bathophenanthroline disulfonate causes immediate inhibition of intracellular primary amine production. A model is proposed in which plasmalemma-bound nitrate reductase reduces extracellular electron acceptors and intracellular nitrate and also acts as a trans-plasmalemma proton pump.