First Observation of Unicellular Organisms Concentrating Arsenic in ACC Intracellular Inclusions in Lake Waters

In unicellular organisms, intracellular inclusions of amorphous calcium carbonate (ACC) were initially described in cyanobacteria and, later, in unicellular eukaryotes from Lake Geneva (Switzerland/France). Inclusions in unicellular eukaryotes, named micropearls, consist of hydrated ACCs, frequently enriched in Sr or Ba, and displaying internal oscillatory zonations, due to variations in the Ba:Ca or Sr:Ca ratios. An analysis of our database, consisting of 1597 micropearl analyses from Lake Geneva and 34 from Lake Titicaca (Bolivia/Peru), showed that a certain number of Sr- and Ba-enriched micropearls from these lakes contain As in amounts measurable by EDXS. A Q-mode statistical analysis confirmed the existence of five chemically distinct morpho-chemical groups of As-bearing micropearls, among which was a new category identified in Lake Geneva, where As is often associated with Mg. This new type of micropearl is possibly produced in a small (7–12 μm size) bi-flagellated organism. Micropearls from Lake Titicaca, which contain Sr, were found in an organism very similar to Tetraselmis cordiformis, which was observed earlier in Lake Geneva. Lake Titicaca micropearls contain larger As amounts, which can be explained by the high As concentration in the water of this lake. The ubiquity of this observed biomineralization process points to the need for a better understanding of the role of amorphous or crystalline calcium carbonates in As cycling in surface waters.

First Observation of Unicellular Organisms Concentrating Arsenic in ACC Intracellular Inclusions

In unicellular organisms, intracellular inclusions of amorphous calcium carbonate (ACC) have been initially described in cyanobacteria and, later, in unicellular eukaryotes of Lake Geneva (Switzerland/France). Inclusions in unicellular eukaryotes ‒named micropearls‒ consist of hydrated ACCs, frequently enriched in Sr or Ba, displaying internal oscillatory zonations due to variations in the Ba:Ca or Sr:Ca ratios. The analysis of our database consisting of 1597 micropearl analyses from Lake Geneva and 34 from Lake Titicaca (Bolivia/Peru) has shown that a certain number of Sr and Ba-enriched micropearls from these lakes contain As in amounts measurable by EDXS. A Q-mode statistical analysis has confirmed the existence of five geochemically distinct morpho-chemical groups of As-bearing micropearls, among which a new category identified in Lake Geneva, where As is often associated with Mg. This new type of micropearl is possibly produced in a small (7-12 m size) bi-flagellated organism. Micropearls from Lake Titicaca, which contain Sr, are found in an organism very similar to Tetraselmis cordiformis, observed in Lake Geneva. Lake Titicaca micropearls contain higher As concentrations which can be explained by the high As concentration in the water of this lake. The ubiquity of the biomineralization process observed points to the need for better understanding of the role of amorphous or crystalline calcium carbonates in As cycling in surface waters.

Differentiation of Crystal Cells, Gravity-Sensing Cells in the Placozoan Trichoplax adhaerens

Trichoplax adhaerens are simple animals with no nervous system, muscles or body axis. Nevertheless, Trichoplax demonstrate complex behaviors, including responses to the direction of the gravity vector. They have only six somatic cell types, and one of them, crystal cells, has been implicated in gravity reception. Multiple crystal cells are scattered near the rim of the pancake-shaped animal; each contains a cup-shaped nucleus and an intracellular crystal, which aligns its position according to the gravity force. Little is known about the development of any cell type in Trichoplax, which, in the laboratory, propagate exclusively by binary fission. Electron and light microscopy were used to investigate the stages by which crystal cells develop their mature phenotypes and distributions. Nascent crystal cells, identified by their possession of a small crystal, were located farther from the rim than mature crystal cells, indicating that crystal cells undergo displacement during maturation. They were elongated in shape and their nucleus was rounded. The crystal develops inside a vacuole flanked by multiple mitochondria, which, perhaps, supply molecules needed for the biomineralization process underlying crystal formation. This research sheds light on the development of unique cells with internal biomineralization and poses questions for further research.

A new gene family diagnostic for intracellular biomineralization of amorphous Ca-carbonates by cyanobacteria

Cyanobacteria have massively contributed to carbonate deposit formation over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein (calcyanin) family specifically associated with cyanobacterial iACC biomineralization. Calcyanin is composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N- terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologues. Calcyanin lacks detectable full-length homologs with known function. Yet, genetic and comparative genomic analyses suggest a possible involvement in Ca homeostasis, making this gene family a particularly interesting target for future functional studies. Whatever its function, this new gene family appears as a gene diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC. This significantly extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Phylogenetic analyses indicate that iACC biomineralization is ancient, with independent losses in various lineages and some HGT cases that resulted in the broad but patchy distribution of calcyanin across modern cyanobacteria. Overall, iACC biomineralization emerges as a new case of genetically controlled biomineralization in bacteria.

Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of β-galactosidase

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from β-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected β-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

Fossil Corals With Various Degrees of Preservation Can Retain Information About Biomineralization-Related Organic Material

Scleractinian corals typically form a robust calcium carbonate skeleton beneath their living tissue. This skeleton, through its trace element composition and isotope ratios, may record environmental conditions of water surrounding the coral animal. While bulk unrecrystallized aragonite coral skeletons can be used to reconstruct past ocean conditions, corals that have undergone significant diagenesis have altered geochemical signatures and are typically assumed to retain insufficient meaningful information for bulk or macrostructural analysis. However, partially recrystallized skeletons may retain organic molecular components of the skeletal organic matrix (SOM), which is secreted by the animal and directs aspects of the biomineralization process. Some SOM proteins can be retained in fossil corals and can potentially provide past oceanographic, ecological, and indirect genetic information. Here, we describe a dataset of scleractinian coral skeletons, aged from modern to Cretaceous plus a Carboniferous rugosan, characterized for their crystallography, trace element composition, and amino acid compositions. We show that some specimens that are partially recrystallized to calcite yield potentially useful biochemical information whereas complete recrystalization or silicification leads to significant alteration or loss of the SOM fraction. Our analysis is informative to biochemical-paleoceanographers as it suggests that previously discounted partially recrystallized coral skeletons may indeed still be useful at the microstructural level.

Uranium Distribution and Incorporation Mechanism in Deep-Sea Corals: Implications for Seawater [CO32–] Proxies

A conservative element in seawater, uranium is readily incorporated into the aragonitic skeletons of scleractinian corals, making them an important paleoclimate archive that can be absolutely dated with U-Th techniques. In addition, uranium concentrations (U/Ca ratios) in corals have been suggested to be influenced by the temperature and/or carbonate ion concentration of the ambient seawater based on empirical calibrations. Microsampling techniques have revealed strong heterogeneities in U/Ca within individual specimens in both surface and deep-sea corals, suggesting a biological control on the U incorporation into the skeletons. Here we further explore the mechanism of uranium incorporation in coral skeletons with the deep-sea species Desmophyllum dianthus, an ideal test organism for the biomineralization processes due to its relatively constant growth environment. We find a negative correlation between bulk coral U/Ca and temperature as well as ambient pH and [CO32–] that is consistent with previous studies. By sampling the growth bands of individual corals, we also find a twofold change in U/Ca within individual corals that is strongly correlated with the δ18O, δ13C, and other Me/Ca ratios of the bands. A similar correlation between U/Ca and stable isotopes as well as other Me/Ca ratios are observed in bulk deep-sea coral samples. With a numerical coral calcification model, we interpret the U/Ca-stable isotope correlation as a result of changes in uranium speciation in response to internal pH elevations in the extracellular calcifying fluid (ECF) of the corals, and suggest that the Ca2UO2(CO3)3(aq) complex, the dominant U species in seawater, may be the major species incorporated into the coral skeleton. Therefore, the correlation between U/Ca and ambient [CO32–] is likely a result of the response of the biomineralization process, especially the magnitude of internal pH elevation, to the growth environment of the corals. Our data suggest overall lower alkalinity pump rates in corals from low saturation seawater compared to those from high saturation seawater, and possible increases in Ca2+ supply from active pumping relative to seawater transport in response to the environmental stress of low saturation.

Environmentally Driven Color Variation in the Pearl Oyster Pinctada margaritifera var. cumingii (Linnaeus, 1758) Is Associated With Differential Methylation of CpGs in Pigment- and Biomineralization-Related Genes

Today, it is common knowledge that environmental factors can change the color of many animals. Studies have shown that the molecular mechanisms underlying such modifications could involve epigenetic factors. Since 2013, the pearl oyster Pinctada margaritifera var. cumingii has become a biological model for questions on color expression and variation in Mollusca. A previous study reported color plasticity in response to water depth variation, specifically a general darkening of the nacre color at greater depth. However, the molecular mechanisms behind this plasticity are still unknown. In this paper, we investigate the possible implication of epigenetic factors controlling shell color variation through a depth variation experiment associated with a DNA methylation study performed at the whole genome level with a constant genetic background. Our results revealed six genes presenting differentially methylated CpGs in response to the environmental change, among which four are linked to pigmentation processes or regulations (GART, ABCC1, MAPKAP1, and GRL101), especially those leading to darker phenotypes. Interestingly, the genes perlucin and MGAT1, both involved in the biomineralization process (deposition of aragonite and calcite crystals), also showed differential methylation, suggesting that a possible difference in the physical/spatial organization of the crystals could cause darkening (iridescence or transparency modification of the biomineral). These findings are of great interest for the pearl production industry, since wholly black pearls and their opposite, the palest pearls, command a higher value on several markets. They also open the route of epigenetic improvement as a new means for pearl production improvement.

An unsuspected biomineralization process in the green algae class Chlorodendrophyceae

<p>Chlorodendrophyceae are a class of unicellular green algae widespread in the aquatic environment (seawater, brackish water, and freshwater) that have recently been discovered to form intracellular carbonates. These mineral inclusions, called <em>micropearls</em>, are mainly composed of hydrated amorphous calcium carbonates (ACC) in which strontium can also accumulate at high concentrations. Under natural and culture conditions, the Sr/Ca ratio of micropearls can be 200 times higher than in their environment, suggesting that Chlorodendrophyceae species may be considered as potential candidates for new bioremediation methods regarding radioactive <sup>90</sup>Sr water contamination. Because very little is known about this phenomenon, ongoing experiments with laboratory cultures are providing essential information about the cellular mechanisms involved in this newly discovered biomineralization process and its impact on the geochemical cycles of Ca and Sr.</p>

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit

The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.