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Unlike most higher plants, unicellular algae can acclimate to changes in irradiance on time scales of hours to a few days. The process involves an enigmatic signaling pathway originating in the plastid that leads to coordinated changes in plastid and nuclear gene expression. To deepen our understanding of this process, we conducted functional studies to examine how the model diatom, Phaeodactylum tricornutum, acclimates to low light and sought to identify the molecules responsible for the phenomenon. We show that two transformants with altered expression of two putative signal transduction molecules, a light-specific soluble kinase and a plastid transmembrane protein, that appears to be regulated by a long noncoding natural antisense transcript, arising from the opposite strand, are physiologically incapable of photoacclimation. Based on these results, we propose a working model of the retrograde feedback in the signaling and regulation of photoacclimation in a marine diatom.

Unicellular photoautotrophs adapt to variations in light intensity by changing the abundance of light harvest pigment-protein complexes (LHCs) on time scales of hours to days. This process requires a feedback signal between the plastid (where light intensity is sensed) to the nucleus (where the genes for LHCs are encoded). The signals must include heretofore unidentified transcription factors that modify the expression level of the LHCs. Analysis of the nuclear genome of the model diatom Phaeodactylum tricornutum revealed that all the lhc genes have potential binding sites for transcription factors belonging to the MYB-family proteins. Functional studies involving antisense RNA interference of a hypothetical protein with a MYB DNA-binding domain were performed. The resultant strains with altered photosynthetic and physiological characteristics lost their ability to acclimate to changes in irradiance; i.e., cellular chlorophyll content became independent of growth irradiance. Our results strongly suggest that the inter-organellar signaling cascade was disrupted, and the cell could no longer communicate the environmental signal from the plastid to the nucleus. Here, we identify, for the first time, an LHC Regulating Myb (LRM) transcription factor, which we propose is involved in lhc gene regulation and photoacclimation mechanisms in response to changes in light intensity.

Sulfur is an essential element of life that is assimilated by Earth’s biosphere through the chemical breakdown of pyrite. On the early Earth, pyrite weathering by atmospheric oxygen was severely limited, and low marine sulfate concentrations persisted for much of the Archean eon. Here, we show an anoxic photochemical mechanism of pyrite weathering that could have provided substantial amounts of sulfate to the oceans as continents formed in the late Archean. Pyrite grains suspended in anoxic ferrous iron solutions produced millimolar sulfate concentrations when irradiated with ultraviolet light. The Fe 2+ (aq) was photooxidized, which, in turn, led to the chemical oxidation of pyritic sulfur. Additional experiments conducted with 2.68 Ga shale demonstrated that photochemically derived ferric iron oxidizes and dissolves sedimentary pyrite during chemical weathering. The results suggest that before the rise of atmospheric oxygen, oxidative pyrite weathering on Archean continents was controlled by the exposure of land to sunlight.

In a rapidly warming world, we ask, "What limits the potential of marine diatoms to acclimate to elevated temperatures?," a group of ecologically successful unicellular eukaryotic photoautotrophs that evolved in a cooler ocean and are critical to marine food webs. To this end, we examined thermal tolerance mechanisms related to photosynthesis in the sequenced and transformable model diatom Phaeodactylum tricornutum. Data from transmission electron microscopy (TEM) and fatty acid methyl ester-gas chromatography mass spectrometry (FAME-GCMS) suggest that saturating thylakoid-associated fatty acids allowed rapid (on the order of hours) thermal tolerance up to 28.5°C. Beyond this critical temperature, thylakoid ultrastructure became severely perturbed. Biophysical analyses revealed that electrochemical leakage through the thylakoid membranes was extremely sensitive to elevated temperature (Q10 of 3.5). Data suggest that the loss of the proton motive force (pmf) occurred even when heat-labile photosystem II (PSII) was functioning, and saturation of thylakoid-associated fatty acids was active. Indeed, growth was inhibited when leakage of pmf through thylakoid membranes was insufficiently compensated by proton input from PSII. Our findings provide a mechanistic understanding of the importance of rapid saturation of thylakoid-associated fatty acids for ultrastructure maintenance and a generation of pmf at elevated temperatures. To the extent these experimental results apply, the ability of diatoms to generate a pmf may be a sensitive parameter for thermal sensitivity diagnosis in phytoplankton.

The most ancient processes for energy production in the evolution of life involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13 amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe]-hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase (ACS), two ancient, extant proteins central to metabolism. These experimental results clearly demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.One Sentence SummarySmall metal-binding peptides were the likely precursors of modern enzymes.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO2, is competitively displaced by O2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO2 for O2. Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO2 and O2. Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO2. This paper identifies a key structural feature that enabled the enzyme to evolve improved CO2 sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.

Significance When oxygenic photosynthesis evolved is debated with an uncertainty of approximately 1 Gy. It is generally assumed that the oxidation of manganese minerals requires biological catalysis or molecular oxygen and therefore is often used as a proxy for the presence of oxygenic photosynthetic organisms. We show that anoxic, abiotic oxidation of the mineral rhodochrosite (MnCO 3 ) by UV light forms H 2 and manganite (γ-MnOOH). Our results reveal an alternative mechanism for producing manganese oxides from rhodochrosite in the absence of molecular oxygen. These results demonstrate the potential impact of photogeochemical processes on the redox state of transition metals and hence question the interpretation of the rise of atmospheric oxygen based on the oxidation of transition metals, such as Cr isotopes.

Phytoplankton are extraordinarily diverse, comprising 13 phylogenetic groups, with diatoms, dinoflagellates, and haptophytes among the most prominent eukaryotes in the ocean. Development of sensor technologies for rapid taxonomic and physiological analysis of phytoplankton communities is crucial for ecological monitoring programs in the global ocean. We describe a novel, ultra-sensitive, multi-spectral fluorescence induction and relaxation instrument (a mini-FIRe) and examine its analytical capability of rapidly determining phytoplankton taxonomic groups, as well as physiological characteristics and photosynthetic rates. We collected and analyzed the database of spectral and photosynthetic properties of major taxonomic groups of phytoplankton. We revealed that the spectral shape of the functional absorption cross-section of Photosystem II (PSII), sPSII(lex), is remarkably constrained within each major phylogenetic group of eukaryotic phytoplankton, including diatoms, haptophytes, dinoflagellates, and chlorophytes. Variability in sPSII(lex) within each group was significantly smaller than the difference between groups. We also examined the classical excitation spectra of chl a fluorescence yields, Fm(lex). Our comparative analysis revealed that sPSII(lex) is a better and more specific proxy for taxonomic analysis. For instance, our developed sPSII-based algorithm correctly identified 90% of experimental data, compared to 77% identified by the Fm-based algorithm. Our results suggest that the multi-color variable fluorescence analysis offers a tool for combined physiological and taxonomic analysis, including identification of major phyla within the ‘red’ lineage of eukaryotic phytoplankton.

Protein coordinated iron-sulfur clusters drive electron flow within metabolic pathways for organisms throughout the tree of life. It is not known how iron-sulfur clusters were first incorporated into proteins. Structural analogies to iron-sulfide minerals present on early Earth, suggest a connection in the evolution of both proteins and minerals. The availability of large protein and mineral crystallographic structure data sets, provides an opportunity to explore co-evolution of proteins and minerals on a large-scale using informatics approaches. However, quantitative comparisons are confounded by the infinite, repeating nature of the mineral lattice, in contrast to metal clusters in proteins, which are finite in size. We address this problem using the Niggli reduction to transform a mineral lattice to a finite, unique structure that when translated reproduces the crystal lattice. Protein and reduced mineral structures were represented as quotient graphs with the edges and nodes corresponding to bonds and atoms, respectively. We developed a graph theory-based method to calculate the maximum common connected edge subgraph (MCCES) between mineral and protein quotient graphs. MCCES can accommodate differences in structural volumes and easily allows additional chemical criteria to be considered when calculating similarity. To account for graph size differences, we use the Tversky similarity index. Using consistent criteria, we found little similarity between putative ancient iron-sulfur protein clusters and iron-sulfur mineral lattices, suggesting these metal sites are not as evolutionarily connected as once thought. We discuss possible evolutionary implications of these findings in addition to suggesting an alternative proxy, mineral surfaces, for better understanding the coevolution of the geosphere and biosphere.

Significance Early life is thought to have evolved from simple building blocks that were propagated through gene duplication events. A classic example is the small soluble iron-sulfur containing protein, bacterial ferredoxin, which is an asymmetric dimer, an essential component of many extant electron transfer chains and has ancient origins. To probe the theoretical gene duplication origins of bacterial ferredoxins, we designed a series of synthetic symmetric constructs. All designs bound two iron-sulfur clusters and were able to support electron transfer between a pair of oxidoreductases in vivo in Escherichia coli . Our results strongly suggest that simple, symmetric ancestral proteins probably evolved early in Earth’s history and can be engineered to facilitate functional electron transfer in synthetic metabolic pathways.

Coral skeletons are materials composed of inorganic aragonitic fibres and organic molecules including proteins, sugars and lipids that are highly organized to form a solid biomaterial upon which the animals live. The skeleton contains tens of proteins, all of which are encoded in the animal genome and secreted during the biomineralization process. While recent advances are revealing the functions and evolutionary history of some of these proteins, how they are spatially arranged in the skeleton is unknown. Using a combination of chemical cross-linking and high-resolution tandem mass spectrometry, we identify, for the first time, the spatial interactions of the proteins embedded within the skeleton of the stony coralStylophora pistillata. Our subsequent network analysis revealed that several coral acid-rich proteins are invariably associated with carbonic anhydrase(s), alpha-collagen, cadherins and other calcium-binding proteins. These spatial arrangements clearly show that protein–protein interactions in coral skeletons are highly coordinated and are key to understanding the formation and persistence of coral skeletons through time.

Life on Earth is driven by electron transfer reactions catalyzed by a suite of enzymes that comprise the superfamily of oxidoreductases (Enzyme Classification EC1). Most modern oxidoreductases are complex in their structure and chemistry and must have evolved from a small set of ancient folds. Ancient oxidoreductases from the Archean Eon between ca. 3.5 and 2.5 billion years ago have been long extinct, making it challenging to retrace evolution by sequence-based phylogeny or ancestral sequence reconstruction. However, three-dimensional topologies of proteins change more slowly than sequences. Using comparative structure and sequence profile-profile alignments, we quantify the similarity between proximal cofactor-binding folds and show that they are derived from a common ancestor. We discovered that two recurring folds were central to the origin of metabolism: ferredoxin and Rossmann-like folds. In turn, these two folds likely shared a common ancestor that, through duplication, recruitment, and diversification, evolved to facilitate electron transfer and catalysis at a very early stage in the origin of metabolism.

The West Antarctic Peninsula (WAP) is a highly productive polar ecosystem where phytoplankton dynamics are regulated by intense bottom‐up control from light and iron availability. Rapid climate change along the WAP is driving shifts in the mixed layer depth and iron availability. Elucidating the relative role of each of these controls and their interactions is crucial for understanding of how primary productivity will change in coming decades. Using a combination of ultra‐high‐resolution variable chlorophyll fluorescence together with fluorescence lifetime analyses on the 2017 Palmer Long Term Ecological Research cruise, we mapped the temporal and spatial variability in phytoplankton photophysiology across the WAP. Highest photosynthetic energy conversion efficiencies and lowest fluorescence quantum yields were observed in iron replete coastal regions. Photosynthetic energy conversion efficiencies decreased by ~ 60% with a proportional increase in quantum yields of thermal dissipation and fluorescence on the outer continental shelf and slope. The combined analysis of variable fluorescence and lifetimes revealed that, in addition to the decrease in the fraction of inactive reaction centers, up to 20% of light harvesting chlorophyll‐protein antenna complexes were energetically uncoupled from photosystem II reaction centers in iron‐limited phytoplankton. These biophysical signatures strongly suggest severe iron limitation of photosynthesis in the surface waters along the continental slope of the WAP.

The reduction of elemental sulfur is an important energy-conserving pathway in prokaryotes inhabiting geothermal environments, where sulfur respiration contributes to sulfur biogeochemical cycling. Despite this, the pathways through which elemental sulfur is reduced to hydrogen sulfide remain unclear in most microorganisms. We integrated growth experiments using Thermovibrio ammonificans, a deep-sea vent thermophile that conserves energy from the oxidation of hydrogen and reduction of both nitrate and elemental sulfur, with comparative transcriptomic and proteomic approaches, coupled with scanning electron microscopy. Our results revealed that two members of the FAD-dependent pyridine nucleotide disulfide reductase family, similar to sulfide-quinone reductase and to NADH-dependent sulfur reductase (NSR), respectively, are over-expressed during sulfur respiration. Scanning electron micrographs and sulfur sequestration experiments indicated that direct access of T. ammonificans to sulfur particles strongly promoted growth. The sulfur metabolism of T. ammonificans appears to require abiotic transition from bulk elemental sulfur to polysulfide to nanoparticulate sulfur at an acidic pH, coupled to biological hydrogen oxidation. A coupled biotic-abiotic mechanism for sulfur respiration is put forward, mediated by an NSR-like protein as the terminal reductase.

Differences in maximal yields of chlorophyll variable fluorescence (Fm) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling Fm above and beyond the QA redox level. Fm was low when the QB binding site was occupied by PQ and high when the QB binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, Fm was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, Fm was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the QB binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize QA–.

Oxidoreductases catalyze electron transfer reactions that ultimately provide the energy for life. A limited set of ancestral protein-metal modules are presumably the building blocks that evolved into this diverse protein family. However, the identity of these modules and their path to modern oxidoreductases is unknown. Using a comparative structural analysis approach, we identify a set of fundamental electron transfer modules that have evolved to form the extant oxidoreductases. Using transition metal-containing cofactors as fiducial markers, it is possible to cluster cofactor microenvironments into as few as four major modules: bacterial ferredoxin, cytochrome c, symerythrin, and plastocyanin-type folds. From structural alignments, it is challenging to ascertain whether modules evolved from a single common ancestor (homology) or arose by independent convergence on a limited set of structural forms (analogy). Additional insight into common origins is contained in the spatial adjacency network (SPAN), which is based on proximity of modules in oxidoreductases containing multiple cofactor electron transfer chains. Electron transfer chains within complex modern oxidoreductases likely evolved through repeated duplication and diversification of ancient modular units that arose in the Archean eon.

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.

Life is based on energy gained by electron-transfer processes; these processes rely on oxidoreductase enzymes, which often contain transition metals in their structures. The availability of different metals and substrates has changed over the course of Earth’s history as a result of secular changes in redox conditions, particularly global oxygenation. New metabolic pathways using different transition metals co-evolved alongside changing redox conditions. Sulfur reduction, sulfate reduction, metha- nogenesis and anoxygenic photosynthesis appeared between about 3.8 and 3.4 billion years ago. The oxidoreductases respon- sible for these metabolisms incorporated metals that were readily available in Archaean oceans, chiefly iron and iron–sulfur clusters. Oxygenic photosynthesis appeared between 3.2 and 2.5 billion years ago, as did methane oxidation, nitrogen fixation, nitrification and denitrification. These metabolisms rely on an expanded range of transition metals presumably made available by the build-up of molecular oxygen in soil crusts and marine microbial mats. The appropriation of copper in enzymes before the Great Oxidation Event is particularly important, as copper is key to nitrogen and methane cycling and was later incorporated into numerous aerobic metabolisms. We find that the diversity of metals used in oxidoreductases has increased through time, suggesting that surface redox potential and metal incorporation influenced the evolution of metabolism, biological electron transfer and microbial ecology.

Understanding the cellular and molecular responses of stony corals to ocean acidification is key to predicting their ability to calcify under projected high CO2 conditions. Of specific interest are the links between biomineralization proteins and the precipitation of new calcium carbonate (CaCO3), which potentially can provide a better understanding of the biomineralization process. We have assessed the effects of increased CO2 on the calcification process in cell cultures of the stony coral, Stylophora pistillata, reared in nutrient-enriched artificial seawater at four pCO2 levels and two glucose concentrations. Dispersed S. pistillata cells grown at low (400 ppmV) and moderate (700 ppmV) pCO2 re-aggregate into proto-polyps and precipitate CaCO3. When grown at pCO2 levels of 1000 ppmV and 2000 ppmV, the cells up-regulate genes for two highly acidic proteins as well as a carbonic anhydrase, but down-regulate long term cadherin protein production and minimize proto-polyp formation, and exhibit a significant decrease in measurable CaCO3 precipitation. However, cell cultures precipitate CaCO3 in all treatments, even at slightly undersaturated conditions (Ωaragonite

Little is known about how stony corals build their calcareous skeletons. There are two prevailing hypotheses: that it is a physicochemically dominated process and that it is a biologically mediated one. Using a combination of ultrahigh-resolution three-dimensional imaging and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, we show that mineral deposition is biologically driven. Randomly arranged, amorphous nanoparticles are initially deposited in microenvironments enriched in organic material; they then aggregate and form ordered aragonitic structures through crystal growth by particle attachment. Our NMR results are consistent with heterogeneous nucleation of the solid mineral phase driven by coral acid-rich proteins. Such a mechanism suggests that stony corals may be able to sustain calcification even under lower pH conditions that do not favor the inorganic precipitation of aragonite.

Amundsen Sea polynyas are among the most productive, yet climate-sensitive ecosystems in the Southern Ocean and host massive annual phytoplankton blooms. These blooms are believed to be controlled by iron fluxes from melting ice and icebergs and by intrusion of nutrient-rich Circumpolar Deep Water, however the interplay between iron effects and other controls, such as light availability, has not yet been quantified. Here, we examine phytoplankton photophysiology in relation to Fe stress and physical forcing in two largest polynyas, Amundsen Sea Polynya (ASP) and Pine Island Polynya (PIP), using the combination of high-resolution variable fluorescence measurements, fluorescence lifetime analysis, photosynthetic rates, and Fe-enrichment incubations. These analyses revealed strong Fe stress in the ASP, whereas the PIP showed virtually no signatures of Fe limitation. In spite of enhanced iron availability in the PIP, chlorophyll biomass remained ∼ 30–50% lower than in the Fe-stressed ASP. This apparent paradox would not have been observed if iron were the main control of phytoplankton bloom in the Amundsen Sea. Long-term satellite-based climatology records revealed that the ASP is exposed to significantly higher solar irradiance levels throughout the summer season, as compared to the PIP region, suggesting that light availability controls the magnitude of phytoplankton blooms in the Amundsen Sea. Our data suggests that higher Fe availability (e.g., due to higher melting rates of ice sheets) would not necessarily increase primary productivity in this region. Furthermore, stronger wind-driven vertical mixing in expanding ice-free areas may lead to reduction in light availability and productivity in the future.

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl-CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50- to 100-fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30- to 50-fold. More important, these cells had a 2-fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using 13 C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl-CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.

We measured the light dependence of gross photosynthesis and oxygen uptake rates by membrane inlet mass spectrometry in two open ocean regions: the Amundsen Sea (Antarctica), dominated by Phaeocystis antarctica, and the North Atlantic, dominated by Emiliania huxleyi. In the North Atlantic, respiration was independent of irradiance and was higher than the gross photosynthetic rate at all irradiances. In contrast, in the Amundsen Sea, oxygen uptake processes were light dependent; dark respiration was one order of magnitude lower than the maximal gross photosynthetic rate, but the oxygen uptake rate increased by 10 fold at surface irradiances. Our results suggest the light dependence of oxygen uptake in Amundsen Sea has two sources: one is independent of photosynthesis, and is possibly associated with the photo-reduction of O2 mediated by dissolved organic matter; the second reflects the activity of an oxidase fueled in the light with photosynthetic electron flow. Our results highlight the importance of improving our understanding of oxygen consuming reactions in the euphotic zone.

Digging into nonclassical pathways to crystallization to unearth design principles for fabricating advanced functionalized materials shapes the future of materials science. Nature has long since been exploiting such nonclassical pathways to crystallization to build inorganic-organic hybrid materials that fulfill support, mastication, defense, attack, or optical functions. Especially, various biomineralizing taxa such as stony corals deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further transform into higher-order mineral structures. Here we examine whether a similar process can be duplicate in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of ultrahigh-resolution imaging, and, in situ, solidstate nuclear magnetic resonance (NMR) spectroscopy, we reveal the underlying mechanism of the phase transformation of these synthetic amorphous nanoparticles into crystals. When soaked in water, these synthetic amorphous nanoparticles are coated by a rigid hydration layer of bound water molecules. In addition, fast chemical exchanges occur between hydrogens from the nanoparticles and those from the free water molecules of the surrounding aqueous medium. At some stage, crystallization spontaneously occurs, and we provide spectroscopic evidence for a solid-state phase transformation of the starting amorphous nanoparticles into crystals. Depending on their initial chemical composition, and especially on the amount of magnesium, the starting amorphous nanoparticles can aggregate and form ordered mineral structures through crystal growth by particle attachment, or rather dissolve and reprecipitate into another crystalline phase. The former scenario offers promising prospects for exerting some control over such non-classical pathway to crystallization to design mineral structures that could not be achieved through a classical layer-by-layer growth.

All life on Earth is dependent on biologically mediated electron transfer (i.e., redox) reactions that are far from thermodynamic equilibrium. Biological redox reactions originally evolved in prokaryotes and ultimately, over the first ∼2.5 billion years of Earth's history, formed a global electronic circuit. To maintain the circuit on a global scale requires that oxidants and reductants be transported; the two major planetary wires that connect global metabolism are geophysical fluids—the atmosphere and the oceans. Because all organisms exchange gases with the environment, the evolution of redox reactions has been a major force in modifying the chemistry at Earth's surface. Here we briefly review the discovery and consequences of redox reactions in microbes with a specific focus on the coevolution of life and geochemical phenomena.

Using solar energy suboptimally How efficient are phytoplankton at converting sunlight into the products of photosynthesis? The two other pathways that that absorbed energy can take are emission back to the environment by fluorescence or conversion to heat. Lin et al. measured phytoplankton fluorescence lifetimes in the laboratory and combined them with satellite measurements of variable chlorophyll fluorescence. Combined, they determined the quantum yields of photochemistry and fluorescence in four ocean basins. Approximately 60% of absorbed solar energy is converted to heat, a figure 50% higher than has been determined for conditions of optimal growth. Science , this issue p. 264

Growth of heterotrophic microbes requires the extraction of energy, electrons, carbon, and nutrients from a complex and dynamic reservoir of potential substrates. We employed a matrix of selected organic substrates with varying characteristics, and experimentally followed the kinetics of assimilation and respiration to explore the basic principles that govern selection and preferential use based on carbon, nitrogen, and energy content. We further competed these substrates in a combinatorial fashion to evaluate preferential substrate utilization in natural microbial assemblages. Several substrates displayed biphasic kinetic responses and variable respiration: assimilation ratios. Amino acids had the shortest turnover times and were taken up preferentially at ambient concentrations. We also identified a linear relationship between substrate uptake rates and affinity, suggesting that the microbial community optimizes the relative abundances of membrane transporters according to substrate demand. When competed against one another at saturating concentrations, substrate assimilation and respiration rates were enhanced or inhibited by up to two orders of magnitude, compared to competitor-free controls. Further, we describe an unexpected trend between the substrate energy density and turnover times, with more energetic, reduced carbon substrates turning over more slowly than more oxidized substrates.

6 pages, 4 figures, supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412883112/-/DCSupplemental, Marine diatoms are silica-precipitating microalgae that account for over half of organic carbon burial in marine sediments and thus they play a key role in the global carbon cycle. Their evolutionary expansion during the Cenozoic era (66 Ma to present) has been associated with a superior competitive ability for silicic acid relative to other siliceous plankton such as radiolarians, which evolved by reducing the weight of their silica test. Here we use a mathematical model in which diatoms and radiolarians compete for silicic acid to show that the observed reduction in the weight of radiolarian tests is insufficient to explain the rise of diatoms. Using the lithium isotope record of seawater as a proxy of silicate rock weathering and erosion, we calculate changes in the input flux of silicic acid to the oceans. Our results indicate that the long-term massive erosion of continental silicates was critical to the subsequent success of diatoms in marine ecosystems over the last 40 My and suggest an increase in the strength and efficiency of the oceanic biological pump over this period, P.C. and S.M.V. are supported by Ramon y Cajal contracts from the Spanish Government. This work was supported by Grants 10PXIB312058-PR from Xunta de Galicia and CTM2011-25035 from the Spanish Ministry of Economy and Competitiveness

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability. Refereed/Peer-reviewed

We examined the effect of cell cycle arrest in the diatom Phaeodactylum tricornutum. When the cycle is disrupted in G1 phase, it leads to unbalanced growth and the accumulation of storage products, especially lipids. In contrast to nitrogen-stressed cells, however, cells arrested in G1 do not cannibalize photosynthetic proteins and show little change in photosynthetic energy conversion efficiency. This study provides insight into how intermediate metabolism is scheduled with respect to the cell cycle in a marine diatom.

Summary Calcium carbonate platforms produced by reef-building stony corals over geologic time are pervasive features around the world [1]; however, the mechanism by which these organisms produce the mineral is poorly understood (see review by [2]). It is generally assumed that stony corals precipitate calcium carbonate extracellularly as aragonite in a calcifying medium between the calicoblastic ectoderm and pre-existing skeleton, separated from the overlying seawater [2]. The calicoblastic ectoderm produces extracellular matrix (ECM) proteins, secreted to the calcifying medium [3–6], which appear to provide the nucleation, alteration, elongation, and inhibition mechanisms of the biomineral [7] and remain occluded and preserved in the skeleton [8–10]. Here we show in cell cultures of the stony coral Stylophora pistillata that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. Analysis of the growing crystals by lattice light-sheet microscopy suggests that the crystals elongate from the cells' surfaces outward.

It is widely established that nutritional nitrogen deprivation increases lipid accumulation but severely decreases growth rate in microalgae. To understand the regulatory branch points that determine the partitioning of carbon among its potential sinks, we analyzed metabolite and transcript levels of central carbon metabolic pathways and determined the average fluxes and quantum requirements for the synthesis of protein, carbohydrates and fatty acid in the diatom Phaeodactylum tricornutum. Under nitrate-starved conditions, the carbon fluxes into all major sinks decrease sharply; the largest decrease was into proteins and smallest was into lipids. This reduction of carbon flux into lipids together with a significantly lower growth rate is responsible for lower overall FA productivities implying that nitrogen starvation is not a bioenergetically feasible strategy for increasing biodiesel production. The reduction in these fluxes was accompanied by an 18-fold increase in a-ketoglutarate (AKG), 3-fold increase in NADPH/NADP þ , and sharp decreases in glutamate (GLU) and glutamine (GLN) levels. Additionally, the mRNA level of acetyl-CoA carboxylase and two type II diacylglycerol-acyltransferases were increased. Partial suppression of nitrate reductase by tungstate resulted in similar trends at lower levels as for nitrate starvation. These results reveal that the GS/GOGAT pathway is the main regulation site for nitrate dependent control of carbon partitioning between protein and lipid biosynthesis, while the AKG/GL(N/U) metabolite ratio is a transcriptional signal, possibly related to redox poise of intermediates in the photosynthetic electron transport system.

Diatoms, unicellular phytoplankton that account for ∼40% of marine primary productivity, often dominate coastal and open-ocean upwelling zones. Limitation of growth and productivity by iron at low light is attributed to an elevated cellular Fe requirement for the synthesis of Fe-rich photosynthetic proteins. In the dynamic coastal environment, Fe concentrations and daily surface irradiance levels can vary by two to three orders of magnitude on short spatial and temporal scales. Although genome-wide studies are beginning to provide insight into the molecular mechanisms used by diatoms to rapidly respond to such fluxes, their functional role in mediating the Fe stress response remains uncharacterized. Here, we show, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid and enhances growth during acute Fe limitation at subsaturating light by increasing the photosynthetic efficiency of carbon fixation. Clone lines overexpressing TpDSP1 had a lower quantum requirement for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I. Cyclic electron flow is an ATP-producing pathway essential in higher plants and chlorophytes with a heretofore unappreciated role in diatoms. However, cells under replete conditions were characterized as having markedly reduced growth and photosynthetic rates at saturating light, thereby constraining the benefits afforded by overexpression. Widespread distribution of DSP-like sequences in environmental metagenomic and metatranscriptomic datasets highlights the presence and relevance of this protein in natural phytoplankton populations in diverse oceanic regimes.

Structure comparison is widely used to quantify protein relationships. Although there are several approaches to calculate structural similarity, specifying significance thresholds for similarity metrics is difficult due to the inherent likeness of common secondary structure elements. In this study, metal co-factor location is used to assess the biological relevance of structural alignments. The distance between the centroids of bound co-factors adds a chemical and function-relevant constraint to the structural superimposition of two proteins. This additional dimension can be used to define cut-off values for discriminating valid and spurious alignments in large alignment sets. The hypothesis underlying our approach is that metal coordination sites constrain structural evolution, thus revealing functional relationships between distantly related proteins. A comparison of three related nitrogenases shows the sequence and fold constraints imposed on the protein structures up to 18 A away from the centers of their bound metal clusters. Proteins 2014; 82:648–656. © 2013 Wiley Periodicals, Inc.

Owing to the considerable current interest in replacing fossil fuels with solar radiation as a clean, renewable, and secure energy source, light-driven electron transport in natural photosynthetic systems offers a valuable blueprint for conversion of sunlight to useful energy forms. In particular, intracytoplasmic membrane vesicles (chromatophores) from the purple bacterium Rhodospirillum rubrum provide a fully functional and robust photosynthetic apparatus, ideal for biophysical investigations of energy transduction and incorporation into biohybrid photoelectrochemical devices. These vesicular organelles, which arise by invagination of the cytoplasmic membrane, are the sites of the photochemical reaction centers and the light harvesting 1 (LH1) complex. The LH1 protein is responsible for collecting visible and near-IR radiant energy and funneling these excitations to the reaction center for conversion into a transmembrane charge separation. Here, we have investigated the morphology, fluorescence kinetics and photocurrent generation of chromatophores from Rsp. rubrum deposited directly onto gold surfaces in the absence of chemical surface modifications. Atomic force microscopy showed a significant coverage of the gold electrode surface by Rsp. rubrum chromatophores. By in situ fluorescence induction/relaxation measurements, a high retention of the quantum yield of photochemistry was demonstrated in the photoactive films. Chronoamperometric measurements showed that the assembled bioelectrodes were capable of generating sustained photocurrent under white light illumination at 220 mW/cm(2) with a maximum current of 1.5 μA/cm(2), which slowly declines in about 1 week. This study demonstrates the possibility of photoelectrochemical control of robust chromatophore preparations from Rsp. rubrum that paves the way for future incorporation into functional solar cells.

In all aquatic environments, nitrogen cycling within the water column is strongly influenced by oxygen. We hypothesize that the nitrogen isotopic composition (δ 15 N) of organic matter deposited in the sediments is a proxy for the redox state of the water column at the time of deposition. We tested the hypothesis by measuring the bulk sedimentary δ 15 N values in a drill core from the Black Sea, a basin that alternates between oxic, less saline conditions and anoxic, marine conditions on glacial–interglacial time scales. We reconstructed these changes in Black Sea redox conditions using sedimentary δ 15 N, total organic carbon (TOC), total nitrogen (TN), redox-sensitive metals, and micropaleontological data from a deep-sea core (DSDP Site 380). The sedimentary data reveal that during the transitions between oxic and anoxic conditions, δ 15 N values increased relative to the preceding and succeeding quasi-steady-state oxic and anoxic periods. The results indicate that the reciprocal transitional states from anoxic to oxic conditions were accompanied by intense denitrification; during the quasi-stable oxic and anoxic states (characterized by glacial fresh water and interglacial marine conditions) nitrification and complete nitrate utilization, respectively, dominate the nitrogen cycle. While other factors may influence the δ 15 N record, our results support the hypothesis that the variations in nitrogen isotopic composition of organic matter are strongly influenced by changes in redox state in the Black Sea subphotic zone on glacial–interglacial time scales, and can be explained by a relatively simple model describing the effects of oxygen on the microbial processes that drive the nitrogen cycle in marine ecosystems. Our model suggests that the nitrogen isotopic composition of marine sediments, on geological time scales, can be used to reconstruct the redox state of the overlying water column.

SummaryBiomineralization is a widely dispersed and highly regulated but poorly understood process by which organisms precipitate minerals from a wide variety of elements [1]. For many years, it has been hypothesized that the biological precipitation of carbonates is catalyzed by and organized on an extracellular organic matrix containing a suite of proteins, lipids, and polysaccharides [2, 3]. The structures of these molecules, their evolutionary history, and the biophysical mechanisms responsible for calcification remain enigmatic. Despite the recognition that mineralized tissues contain proteins that are unusually rich in aspartic and glutamic acids [4–6], the role of these proteins in biomineralization remains elusive [5, 6]. Here we report, for the first time, the identification, cloning, amino acid sequence, and characterization of four highly acidic proteins, derived from expression of genes obtained from the common stony coral, Stylophora pistillata. Each of these four proteins can spontaneously catalyze the precipitation of calcium carbonate in vitro. Our results demonstrate that coral acid-rich proteins (CARPs) not only bind Ca2+ stoichiometrically but also precipitate aragonite in vitro in seawater at pH 8.2 and 7.6, via an electrostatic interaction with protons on bicarbonate anions. Phylogenetic analysis suggests that at least one of the CARPs arose from a gene fusion. Similar, highly acidic proteins appear to have evolved several times independently in metazoans through convergence. Based purely on thermodynamic grounds, the predicted change in surface ocean pH in the next decades would appear to have minimal effect on the capacity of these acid-rich proteins to precipitate carbonates.

Molybdoenzymes are an ancient protein family found in phylogenetically and ecologically diverse prokaryotes. Under anaerobic conditions, respiratory molybdoenzymes catalyze redox reactions that transfer electrons to a variety of substrates that act as terminal electron acceptors for energy generation. Here, we used probe sequences to conduct an extensive genomic survey and phylogenetic inference for NarG, DmsA, TorA and nine other respiratory molybdoenzyme subfamilies. Our analysis demonstrates their abundance in 60% of prokaryotic phyla. In contrast to many other autonomic genetic units in prokaryotes, the major route of evolution of their predominant subfamilies is vertical gene transfer, gene duplication and divergence. Our results show the robustness of genomic co-occurrence of respiratory molybdoenzymes genes, found in the majority of studied species, for most of the enzyme subfamilies. Genomes which encode for multiple respiratory molybdoenzymes are also enriched in genes regulating replication, recombination and mobility of genetic elements. Respiratory molybdoenzymes were found in prokaryotes associated with diverse environments occupying terrestrial, aquatic, food and host-related habitats, emphasizing their essential role in adaptation of prokaryotes to changing environments. Interestingly, host-associated prokaryotes such as human pathogens more frequently carry multiple respiratory molybdoenzyme genes compared with non-host-associated prokaryotes, highlighting the importance of metabolic flexibility in host-microbiome environments.

Mesodinium rubrum (=Myrionecta rubra), a marine ciliate, acquires plastids, mitochondria, and nuclei from cryptophyte algae. Using a strain of M. rubrum isolated from McMurdo Sound, Antarctica, we investigated the photoacclimation potential of this trophically unique organism at a range of low irradiance levels. The compensation growth irradiance for M. rubrum was 0.5 μmol quanta · m(-2) · s(-1) , and growth rate saturated at ∼20 μmol quanta · m(-2) · s(-1) . The strain displayed trends in photosynthetic efficiency and pigment content characteristic of marine phototrophs. Maximum chl a-specific photosynthetic rates were an order of magnitude slower than temperate strains, while growth rates were half as large, suggesting that a thermal limit to enzyme kinetics produces a fundamental limit to cell function. M. rubrum acclimates to light- and temperature-limited polar conditions and closely regulates photosynthesis in its cryptophyte organelles. By acquiring and maintaining physiologically viable, plastic plastids, M. rubrum establishes a selective advantage over purely heterotrophic ciliates but reduces competition with other phototrophs by exploiting a very low-light niche.