A joint proteomic and genomic investigation provides insights into the mechanism of calcification in coccolithophores.

Coccolithophores are globally abundant, calcifying microalgae that have profound effects on marine biogeochemical cycles, the climate, and life in the oceans. They are characterized by a cell wall of CaCO3 scales called coccoliths, which may contribute to their ecological success. The intricate morphologies of coccoliths are of interest for biomimetic materials synthesis. Despite the global impact of coccolithophore calcification, we know little about the molecular machinery underpinning coccolithophore biology. Working on the model Emiliania huxleyi, a globally distributed bloom-former, we deploy a range of proteomic strategies to identify coccolithogenesis-related proteins. These analyses are supported by a new genome, with gene models derived from long-read transcriptome sequencing, which revealed many novel proteins specific to the calcifying haptophytes. Our experiments provide insights into proteins involved in various aspects of coccolithogenesis. Our improved genome, complemented with transcriptomic and proteomic data, constitutes a new resource for investigating fundamental aspects of coccolithophore biology.

Annotated genome sequences of the carnivorous plant Roridula gorgonias and a non-carnivorous relative, Clethra arborea.

OBJECTIVE: Plant carnivory is distributed across the tree of life and has evolved at least six times independently, but sequenced and annotated nuclear genomes of carnivorous plants are currently lacking. We have sequenced and structurally annotated the nuclear genome of the carnivorous Roridula gorgonias and that of a non-carnivorous relative, Madeira's lily-of-the-valley-tree, Clethra arborea, both within the Ericales. This data adds an important resource to study the evolutionary genetics of plant carnivory across angiosperm lineages and also for functional and systematic aspects of plants within the Ericales. RESULTS: Our assemblies have total lengths of 284 Mbp (R. gorgonias) and 511 Mbp (C. arborea) and show high BUSCO scores of 84.2% and 89.5%, respectively. We used their predicted genes together with publicly available data from other Ericales' genomes and transcriptomes to assemble a phylogenomic data set for the inference of a species tree. However, groups of orthologs showed a marked absence of species represented by a transcriptome. We discuss possible reasons and caution against combining predicted genes from genome- and transriptome-based assemblies.

Surface-Enhanced Raman Scattering Microspectroscopy Enables the Direct Characterization of Biomineral-Associated Organic Material on Single Calcareous Microskeletons.

Biominerals are composite materials with inorganic and organic components. The latter provide insights into how organisms control mineralization and, if derived from micro/nannofossils, into past climates. Many calcifying organisms cannot be cultured or are extinct; the only materials available for their study are therefore complex environmental samples in which the organism of interest may only be a minor component. There is currently no method for characterizing the biomineral-associated organic material from single particles within such assemblages, so its compositional diversity is unknown. Focusing on coccoliths, we demonstrate that surface-enhanced Raman scattering microspectroscopy can be used to determine the origin and composition of fossil organic matter at the single-particle level in a heterogeneous micro/nannofossil assemblage. This approach may find applications in the study of micro/nannofossil assemblages and uncultivated species, providing evolutionary insights into the macromolecular repertoire involved in biomineralization.

An Efficient Method for the Plating of Haploid and Diploid Emiliania huxleyi on Solid Medium1.

Emiliania huxleyi is a globally important coccolithophore and one of the most successful eukaryotic organisms in the modern oceans. Despite a large body of work on this organism, including the sequencing of its genome, the tools required for forward and reverse functional genetic studies are still undeveloped. Here we present an optimized method for the clonal isolation of E. huxleyi by plating on solid medium. We demonstrate the utility of this method for a variety of strains including haploid, calcifying-diploid, and noncalcifying diploid strains. We show that, in contrast to previous studies, no changes in cell ploidy status occur when the cells are plated. Our method will greatly aid attempts to elucidate the genetic basis of the remarkable physiology of E. huxleyi by forward and reverse genetic approaches.

Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles.

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi, raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions.

Exploiting algal mineralization for nanotechnology: bringing coccoliths to the fore.

Complex mineral structures are produced by many microalgal species. Pioneering work on diatom silica has demonstrated the potential of such structures in nanotechnology. The calcified scales of coccolithophores (coccoliths) have received less attention, but the large diversity of architectures make coccoliths attractive as parts for nano-devices. Currently coccolith calcite can be modified by the incorporation of metal ions or adsorption of enzymes to the surface, but genetic modification of coccolithophores may permit the production of coccoliths with customized architectures and surface properties. Further work on the laboratory cultivation of diverse species, the physiochemical properties of coccoliths and on genetic tools for coccolithophores will be necessary to realize the full potential of coccoliths in nanotechnology.

Trace-Element Incorporation into Intracellular Pools Uncovers Calcium-Pathways in a Coccolithophore.

Many organisms form minerals from precursor phases that crystallize under strict biological control. The dynamic intracellular processes of formation, transport, and deposition of these precursor phases are challenging to identify. An unusual situation is recently revealed for the calcifying alga Emiliania huxleyi, as the cells contain a compartment filled with a concentrated Ca and P phase but the final calcite crystals, which are nucleated in a different compartment, are P-free. Thus, the connection of the Ca-P-rich pool to the mineralization process remains unclear. Here, pulse-chase experiments are used with Sr to label the Ca-P-rich phase in E. huxleyi cells, and cryo X-ray absorption spectroscopy and analytical transmission electron microscopy to follow the Sr within cells. It is found that Sr is first found in the Ca-P-rich phase and then becomes incorporated into the calcite. This demonstrates that the calcium used by the cells to build calcite originates from the Ca-P-rich pool.

Templated and self-limiting calcite formation directed by coccolith organic macromolecules.

The formation of intricately shaped crystalline minerals by organisms is orchestrated by specialized biomacromolecules. The macromolecules associated with coccoliths, nanometer-sized calcite crystal arrays produced by marine microalgae, can form a distinct calcium-rich phase via macromolecular recognition. Here, we show that this calcium-rich phase can be mineralized into a thin film of single-crystalline calcite by the balanced addition of carbonate ions. Such a crystallization process provides a strategy to direct crystalline products via local interactions between soluble macromolecules and compatible templates.

Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day-1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate.

Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes.

Marine algae produce a variety of glycans, which fulfill diverse biological functions and fuel the carbon and energy demands of heterotrophic microbes. A common approach to analysis of marine organic matter uses acid to hydrolyze the glycans into measurable monosaccharides. The monosaccharides may be derived from different glycans that are built with the same monosaccharides, however, and this approach does not distinguish between glycans in natural samples. Here we use enzymes to digest selectively and thereby quantify laminarin in particulate organic matter. Environmental metaproteome data revealed carbohydrate-active enzymes from marine flavobacteria as tools for selective hydrolysis of the algal ß-glucan laminarin. The enzymes digested laminarin into glucose and oligosaccharides, which we measured with standard methods to establish the amounts of laminarin in the samples. We cloned, expressed, purified, and characterized three new glycoside hydrolases (GHs) of Formosa bacteria: two are endo-ß-1,3-glucanases, of the GH16 and GH17 families, and the other is a GH30 exo-ß-1,6-glucanase. Formosa sp. nov strain Hel1_33_131 GH30 (FbGH30) removed the ß-1,6-glucose side chains, and Formosa agariphila GH17A (FaGH17A) and FaGH16A hydrolyzed the ß-1,3-glucose backbone of laminarin. Specificity profiling with a library of glucan oligosaccharides and polysaccharides revealed that FaGH17A and FbGH30 were highly specific enzymes, while FaGH16A also hydrolyzed mixed-linked glucans with ß-1,4-glucose. Therefore, we chose the more specific FaGH17A and FbGH30 to quantify laminarin in two cultured diatoms, namely, Thalassiosira weissflogii and Thalassiosira pseudonana, and in seawater samples from the North Sea and the Arctic Ocean. Combined, these results demonstrate the potential of enzymes for faster, stereospecific, and sequence-specific analysis of select glycans in marine organic matter.IMPORTANCE Marine algae synthesize substantial amounts of the glucose polymer laminarin for energy and carbon storage. Its concentrations, rates of production by autotrophic organisms, and rates of digestion by heterotrophic organisms remain unknown. Here we present a method based on enzymes that hydrolyze laminarin and enable its quantification even in crude substrate mixtures, without purification. Compared to the commonly used acid hydrolysis, the enzymatic method presented here is faster and stereospecific and selectively cleaves laminarin in mixtures of glycans, releasing only glucose and oligosaccharides, which can be easily quantified with reducing sugar assays.

Lattice distortions in coccolith calcite crystals originate from occlusion of biomacromolecules.

During biomineralization, organisms control the formation and morphology of a mineral using biomacromolecules. The biomacromolecules that most strongly interact with the growing crystals frequently get occluded within. Such an observation has been recently obtained for the calcium carbonate producing coccolithophore species Pleurochrysis carterae. Coccolithophores are unicellular algae that produce calcified scales built from complex-shaped calcite crystals, termed coccoliths. It is unclear how widespread the phenomenon of biomacromolecular occlusion within calcite crystals is in calcifying haptophytes such as coccolithophores. Here, the coccoliths of biological replicates of the bloom forming Emiliania huxleyi are compared with that of Pleurochrysis carterae, two species with different coccolith morphologies and crystal growth mechanisms. From high-resolution synchrotron X-ray diffraction, changes in the lattice parameters of coccolith calcite, after heating to 450°C, are observed and associated with macrostrain originating from occluded biomacromolecules. We propose a mechanism governing the biomacromolecules' interaction with the growing coccolith crystals and their likely origin.

Macromolecular recognition directs calcium ions to coccolith mineralization sites.

Many organisms form elaborate mineralized structures, constituted of highly organized arrangements of crystals and organic macromolecules. The localization of crystals within these structures is presumably determined by the interaction of nucleating macromolecules with the mineral phase. Here we show that, preceding nucleation, a specific interaction between soluble organic molecules and an organic backbone structure directs mineral components to specific sites. This strategy underlies the formation of coccoliths, which are highly ordered arrangements of calcite crystals produced by marine microalgae. On combining the insoluble organic coccolith scaffold with coccolith-associated soluble macromolecules in vitro, we found a massive accretion of calcium ions at the sites where the crystals form in vivo. The in vitro process exhibits profound similarities to the initial stages of coccolith biogenesis in vivo.

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga.

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana.

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.

Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana.

The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO2 (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12-14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.

Gas-Chromatography Mass-Spectrometry (GC-MS) Based Metabolite Profiling Reveals Mannitol as a Major Storage Carbohydrate in the Coccolithophorid Alga Emiliania huxleyi.

Algae are divergent organisms having a wide variety of evolutional histories. Although most of them share photosynthetic activity, their pathways of primary carbon metabolism are rather diverse among species. Here we developed a method for gas chromatography-mass spectroscopy (GC-MS) based metabolite profiling for the coccolithophorid alga Emiliania huxleyi, which is one of the most abundant microalgae in the ocean, in order to gain an overview of the pathway of primary metabolism within this alga. Following method optimization, twenty-six metabolites could be detected by this method. Whilst most proteogenic amino acids were detected, no peaks corresponding to malate and fumarate were found. The metabolite profile of E. huxleyi was, however, characterized by a prominent accumulation of mannitol reaching in excess of 14 nmol 106 cells-1. Similarly, the accumulation of the 13C label during short term H13CO3- feeding revealed a massive redistribution of label into mannitol as well as rapid but saturating label accumulation into glucose and several amino acids including aspartate, glycine and serine. These results provide support to previous work suggesting that this species adopts C3 photosynthesis and that mannitol functions as a carbon store in E. huxleyi.

Nanopatterned protein microrings from a diatom that direct silica morphogenesis.

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO(2) (silica). Formation of the intricate silica structures of diatoms is regarded as a paradigm for biomolecule-controlled self-assembly of three-dimensional, nano- to microscale-patterned inorganic materials. Silica formation involves long-chain polyamines and phosphoproteins (silaffins and silacidins), which are readily soluble in water, and spontaneously form dynamic supramolecular assemblies that accelerate silica deposition and influence silica morphogenesis in vitro. However, synthesis of diatom-like silica structure in vitro has not yet been accomplished, indicating that additional components are required. Here we describe the discovery and intracellular location of six novel proteins (cingulins) that are integral components of a silica-forming organic matrix (microrings) in the diatom Thalassiosira pseudonana. The cingulin-containing microrings are specifically associated with girdle bands, which constitute a substantial part of diatom biosilica. Remarkably, the microrings exhibit protein-based nanopatterns that closely resemble characteristic features of the girdle band silica nanopatterns. Upon the addition of silicic acid the microrings become rapidly mineralized in vitro generating nanopatterned silica replicas of the microring structures. A silica-forming organic matrix with characteristic nanopatterns was also discovered in the diatom Coscinodiscus wailesii, which suggests that preassembled protein-based templates might be general components of the cellular machinery for silica morphogenesis in diatoms. These data provide fundamentally new insight into the molecular mechanisms of biological silica morphogenesis, and may lead to the development of self-assembled 3D mineral forming protein scaffolds with designed nanopatterns for a host of applications in nanotechnology.

Loss of the actin-like protein MamK has pleiotropic effects on magnetosome formation and chain assembly in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane-enclosed magnetite crystals. For magnetic orientation individual magnetosome particles are assembled into well-organized chains. The actin-like MamK and the acidic MamJ proteins were previously implicated in chain assembly. While MamK was suggested to form magnetosome-associated cytoskeletal filaments, MamJ is assumed to attach the magnetosome vesicles to these structures. Although the deletion of either mamK in Magnetospirillum magneticum, or mamJ in Magnetospirillum gryphiswaldense affected chain formation, the previously observed phenotypes were not fully consistent, suggesting different mechanisms of magnetosome chain assembly in both organisms. Here we show that in M. gryphiswaldense MamK is not absolutely required for chain formation. Straight chains, albeit shorter, fragmented and ectopic, were still formed in a mamK deletion mutant, although magnetosome filaments were absent as shown by cryo-electron tomography. Loss of MamK also resulted in reduced numbers of magnetite crystals and magnetosome vesicles and led to the mislocalization of MamJ. In addition, extensive analysis of wild type and mutant cells revealed previously unidentified ultrastructural characteristics in M. gryphiswaldense. Our results suggest that, despite of their functional equivalence, loss of MamK proteins in different bacteria may result in distinct phenotypes, which might be due to a species-specific genetic context.

The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals.

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe3O4) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.

The acidic repetitive domain of the Magnetospirillum gryphiswaldense MamJ protein displays hypervariability but is not required for magnetosome chain assembly.

Magnetotactic bacteria navigate along the earth's magnetic field using chains of magnetosomes, which are intracellular organelles comprising membrane-enclosed magnetite crystals. The assembly of highly ordered magnetosome chains is under genetic control and involves several specific proteins. Based on genetic and cryo-electron tomography studies, a model was recently proposed in which the acidic MamJ magnetosome protein attaches magnetosome vesicles to the actin-like cytoskeletal filament formed by MamK, thereby preventing magnetosome chains from collapsing. However, the exact functions as well as the mode of interaction between MamK and MamJ are unknown. Here, we demonstrate that several functional MamJ variants from Magnetospirillum gryphiswaldense and other magnetotactic bacteria share an acidic and repetitive central domain, which displays an unusual intra- and interspecies sequence polymorphism, probably caused by homologous recombination between identical copies of Glu- and Pro-rich repeats. Surprisingly, mamJ mutant alleles in which the central domain was deleted retained their potential to restore chain formation in a DeltamamJ mutant, suggesting that the acidic domain is not essential for MamJ's function. Results of two-hybrid experiments indicate that MamJ physically interacts with MamK, and two distinct sequence regions within MamJ were shown to be involved in binding to MamK. Mutant variants of MamJ lacking either of the binding domains were unable to functionally complement the DeltamamJ mutant. In addition, two-hybrid experiments suggest both MamK-binding domains of MamJ confer oligomerization of MamJ. In summary, our data reveal domains required for the functions of the MamJ protein in chain assembly and maintenance and provide the first experimental indications for a direct interaction between MamJ and the cytoskeletal filament protein MamK.