The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

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The oyster genome reveals stress adaptation and complexity of shell formation

Diatom cell walls are regarded as a paradigm for controlled production of nanostructured silica, but the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. A set of polycationic peptides (called silaffins) isolated from diatom cell walls were shown to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Silaffins contain covalently modified lysine-lysine elements. The first lysine bears a polyamine consisting of 6 to 11 repeats of the N-methyl-propylamine unit. The second lysine was identified as epsilon-N,N-dimethyl-lysine. These modifications drastically influence the silica-precipitating activity of silaffins.

http://science.sciencemag.org/content/sci/286/5442/1129.full.pdf

Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation

2.3. Amorphous Minerals 4354 3. Biological Routes to Controlling Morphology 4354 3.1. General Mechanisms 4356 3.1.1. Soluble and Insoluble Organic Molecules 4356 3.1.2. Control over Crystal Polymorph 4357 3.1.3. Control over Crystal Orientation 4359 3.2. Single-Crystal Biominerals 4359 3.2.1. Organic and Inorganic Soluble Additives 4360 3.2.2. Templating of Single-Crystal Morphologies 4361 3.3. Polycrystalline Biominerals 4366 3.3.1. Nacre Formation in Mollusks 4366 3.3.2. ForaminiferasA Biogenic Mesocrystal 4368 3.4. Amorphous Biominerals 4370 3.4.1. Silicification in Diatoms 4370 3.4.2. In Vitro Studies of Silicification in Diatoms 4371 4. Bioinspired Routes to Controlling Crystal Morphologies 4371

Controlling Mineral Morphologies and Structures in Biological and Synthetic Systems

The systematic fabrication of advanced materials will require the construction of architectures over scales ranging from the molecular to the macroscopic. The basic constructional processes of biomineralization—supramolecular pre-organization, interfacial molecular recognition (templating) and cellular processing—can provide useful archetypes for molecular-scale building, or ‘molecular tectonics’, in inorganic materials chemistry.

Molecular tectonics in biomineralization and biomimetic materials chemistry

In the most general sense, biomineralization is a process by which organisms produce materials solutions for their own functional requirements. Because so many biomineral products are derived from an initial solution phase and are either completely crystalline or include crystalline components, an understanding of the physical principles of crystallization from solutions is an important tool for students of biomineralization. However, crystal growth is a science of great breadth and depth, about which many extensive texts have been written. In addition, there are already other thorough reviews that specifically address the crystal growth field of study as it relates to biomineral formation. Consequently, the goals of this chapter are both modest and specific. It is intended to provide: 1) a simple narrative explaining the physical principles behind crystallization for those who are completely new to the topic, 2) a few basic equations governing nucleation and growth for those who wish to apply those principles—at least in a semi-quantitative fashion—to experimental observations of mineralization, and 3) an overview of some recent molecular-scale studies that have revealed new insights into the control of crystal growth by small molecules, both organic and inorganic.

This last topic gets to the heart of what makes crystallization in biological systems unique. Every day, many tons of crystals are produced synthetically in non-biological processes, but by-and-large, the degree of control over nucleation and growth achieved by deterministic additions of growth modifiers or the presence of a controlling matrix is very minor. More commonly, crystal growers view modifying agents as unwanted impurities and work extremely hard to eliminate them from the starting materials. Indeed, the degree to which living organisms are able to control the crystallization process is most striking when contrasted to the products of such synthetic crystallization processes. This contrast applies to both the compositional differences that …

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Principles of crystal nucleation and growth

The growth of molluscan shell crystals is usually thought to be initiated from solution by extracellular organic matrix. We report a class of granulocytic hemocytes that may be directly involved in shell crystal production for oysters. On the basis of scanning electron microscopy (SEM) and x-ray microanalysis, these granulocytes contain calcium carbonate crystals, and they increase in abundance relative to other hemocytes following experimentally induced shell regeneration. Hemocytes are observed at the mineralization front using vital fluorescent staining and SEM. Some cells are observed releasing crystals that are subsequently remodeled, thereby at least augmenting matrix-mediated crystal-forming processes in this system.

https://science.sciencemag.org/content/sci/304/5668/297.full.pdf

Hemocyte-mediated shell mineralization in the eastern oyster.

A calcium-binding soluble protein extracted from oyster shell suppresses calcium carbonate nucleation and decreases the rate of crystal growth in vitro. These findings suggest that soluble matrix may regulate shell growth.

https://science.sciencemag.org/content/sci/212/4501/1397.full.pdf

Control of Calcium Carbonate Nucleation and Crystal Growth by Soluble Matrx of Oyster Shell

The organic matrix of calcium carbonate structures can be involved in the formation of the biomineral by regulating various stages of crystal growth. The preponderance of evidence suggests that matrix can act as an initiator of crystal growth at the mineralizing front. The mechanism for this initiation is not known. However, the matrix may provide a template for the crystal lattice, or more likely, a surface that can simply stabilize the critical nucleus of calcium and carbonate. The degree of specialization of the nucleation sites is uncertain; however, they are hypothesized to be a class of soluble, acidic, calcium-binding polymers which are present in matrix extracted from biomineral. Recent studies have revealed that similar soluble components of extracted matrix have the capability of inhibiting nucleation and crystal growth in vitro and biomineralization in organisms exposed to the extracts. Further, crystals grown in vitro in the presence of extracted matrix have a morphology distinct from those of crystals grown without matrix. These findings generally support the hypotheses generated either a priori or from morphological observations which suggest that matrix must limit crystal growth and may well influence structure of biomineral. Hypotheses which explain a dual function of the soluble matrix have these components periodically released to the mineralization front where they can associate with more insoluble forms of matrix and sequentially initiate or inhibit growth. There also exists evidence to suggest that soluble regulatory components are intra-crystalline in orientation and thus may be released continuously

Regulation of Carbonate Calcification by Organic Matrix

Most biominerals appear to be composites of organic material and mineral. Whether biosilica is such a composite is unresolved because of a lack of evidence for such organic components. We present evidence that organic material exists within diatom biosilica and can be extracted using HF/NH4F solutions from frustules isolated from Cyclotella meneghiniana Kutz and diatomaceous earth. To eliminate organic casing on the silicified frustules as a source of organic materials, the casing was removed by oxidation of frustules with NaOCl before extraction. The removal of the casing was confirmed in that oxidized frustules no longer displayed the ability to be stained with ruthenium red and fluorescamine. Frustules examined with EDXA showed an emission peak from sulfur before treatment but no peak following treatment, indicating that oxidation removed organic sulfur. The organic material obtained from extracts of fresh frustules contained both soluble and insoluble components. Only soluble material was evident in extracts from diatomaceous earth. The soluble material appears to contain glycoproteins with relatively high levels of serine and glycine. The soluble proteins from fresh frustules also appear to be phosphorylated. Indirect evidence is presented that suggests the soluble proteins may contain regions of primary structure enriched in anionic amino acids. The soluble extracts differ from general cell contents when the two fractions are compared, suggesting that frustules contain specialized organic material. The identification of silica-specific organic material suggests that mineralization in diatoms may be in part matrix-mediated.

Evidence of an organic matrix from diatom biosilica1

Correlations between structural properties of bivalve shell organic matrix and its proposed functions in the regulation of biomineralization were examined using proteinaceous fractions obtained from the shell of the oyster Crassostrea virginica (Gmelin) following dissolution of the mineral with ethylenediaminetetraacetate (EDTA). Matrix isolated in this way contains a continuous size distribution of proteins ranging from relatively small molecular weight (Mr) soluble matrix (SM) components to insoluble matrix (IM) components. Regulation of mineralization by these components was determined primarily by their reduction of crystal growth rate in an in vitro assay. For all fractions tested, there was an inverse correlation between Mr and regulatory activity. However, matrix properties other than molecular size may be important in regulation of crystal growth in vivo in that the larger but less acidic of two soluble matrix proteins was a more effective inhibitor of spicule formation by sea urchin embryos. Base treatment of IM and high molecular weight SM fractions that had little or no inhibitory activity when untreated, resulted in constituents that had a molecular weight distribution and in vitro inhibitory activity in the same range as whole SM. These results, combined with the finding that a major fraction of IM has an amino acid composition very similar to the highly charged SM fractions, suggest that much of the matrix is made up of similar molecules, and that their function in crystal growth regulation may change as they interact to form units of increasing size. A second class of IM was isolated which contained dihydroxyphenylalanine (dopa) and a preponderance of hydrophobic amino acids. This material may represent the basic structural framework of the matrix.

A. P. Wheeler

Papers

Hemocyte-mediated shell mineralization in the eastern oyster.

Control of Calcium Carbonate Nucleation and Crystal Growth by Soluble Matrx of Oyster Shell

Regulation of Carbonate Calcification by Organic Matrix

Evidence of an organic matrix from diatom biosilica1

Regulation of in vitro and in vivo CaCO3 crystallization by fractions of oyster shell organic matrix