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A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

Amino Acid Sequence↗

Trichoderma koningii as a biomineralizing fungous agent of calcium oxalate crystals in typical Argiudolls of the Los Padres Lake natural reserve (Buenos Aires, Argentina).

The aim of the present study, performed on typical Argiudolls in a natural reserve with little or no anthropic impact, was to characterize the fungous biomineralizing process of calcium oxalate crystals in organic horizons of the soil. The chosen sites possessed different plant cover, identified as acacia woods and grassy meadows with particular micro environmental conditions that have differing effects in the process of biomineralization. The contribution of the plant material in the soil is a key factor since 1) it generates the particular composition of the organic horizons, 2) it determines the nature of decomposing organisms, and 3) it affects the presence, composition and development of biominerals. According to the results obtained, the acacia woods prove to be a site comparatively more favorable to the fungous biomineralizing process. This makes itself manifest in the greater abundance and development of crystals in the organic horizons of the soil, resulting in whewellite (CaC2O4.H2O) and weddellite (CaC2O4.(2+x) H2O) regarding biomineral species developed, the latter being the major component. The observation of both species of biominerals is noteworthy since it represents the first cited in the country. The isolated fungous organisms were Trichoderma koningii, and Absidia corymbifera. T. koningii was identified as the most active biomineralizing organism thus constituting the first reference to indicate this species as a biomineral producing agent.

Acacia↗

Composition of biomineral organic matrices with special emphasis on turbot (Psetta maxima) otolith and endolymph.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Animals↗

A Functionally Conserved yet Dynamically Evolving Toolkit Underpinning Molluscan Biomineralization: Insights From Shell and Radula.

The molluscan shell and radula constitute pivotal molluscan innovations, each characterized by distinct functions and diverse forms, regulated by the highly specific biomineralization regulatory networks. Despite their paramount importance, the conserved components and adaptive evolutionary processes governing these regulatory networks remain unresolved. To address this knowledge gap, we advocate for the integration of data from less-explored lineages, such as Scaphopoda, as an essential step. This study presents the inaugural comprehensive transcriptome analysis of Pictodentalium vernedei, a representative species of Scaphopoda distinguished by a unique and evolutionarily conserved shell morphology and radula structure. Furthermore, comparative transcriptome/genome analyses are employed to unravel the conservatism and evolutionary innovation of the involved biomineralization regulatory elements. Our findings underscore the central role of secretomes in governing biomineralization processes, and we identified a fundamental set of 26 domains within molluscan secretomes, forming an essential functional protein domain repertoire necessary for the transformation of inorganic ions into biomineralized structures. This core biomineralization toolkit has undergone independent expansion and lineage-specific recruitment, giving rise to novel, modular domain architectures. This may be essential for the functional specialization and morphological diversification of shell and radula structures. These evolutionary processes are driven by the independent co-option of ancient genes and the emergence of novel de novo genes. This comprehensive investigation not only contributes insights into the evolution of molluscan biomineralization structures but also establishes avenues for further scholarly exploration.

Animals↗

Studies of biominerals relevant to the search for life on Mars.

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO(3)) and aragonite, a metastable state of calcite.We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2-25 mum wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin.Obviously, if life existed in the past on the Red Planet, we could expect to find "ancient" biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485 degrees C. Instead, the calcite behaves like abiotic aragonite.Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and "sample-return" Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies.

Calcium Carbonate↗

Influence of microgravity on crystal formation in biomineralization.

Biomineralized tissues are widespread in animals. They are essential elements in skeletons and in statocysts. The function of both can only be understood with respect to gravitational force, which has always been present. Therefore, it is not astonishing to identify microgravity as a factor influencing biomineralization, normally resulting in the reduction of biomineralized materials. All known biominerals are composite materials, in which the organic matrix and the inorganic materials, organized in crystals, interact. If, during remodeling and turnover processes under microgravity, a defective organization of these crystals occurs, a reduction in biomineralized materials could be the result. To understand the influence of microgravity on the formation of biocrystals, we studied the shell-building process of the snail Biomphalaria glabrata as a model system. We show that, under microgravity (space shuttle flights STS-89 and STS-90), shell material is built in a regular way in both adult snails and snail embryos during the beginning of shell development. Microgravity does not influence crystal formation. Because gravity has constantly influenced evolution, the organization of biominerals with densities near 3 must have gained independence from gravitational forces, possibly early in evolution.

Animals↗

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.

Animals↗

Templating and supersaturation-driven anti-templating: principles of biomineral architecture.

The structural synergy between biominerals (CaCO(3), hydroxyapatite) and biosubstrates were examined for the first time. The templating effect of substrate and a newly identified supersaturation-driven interfacial structure mismatch effect were identified in the context of a new nucleation model. It follows that the heterogeneous nucleation which corresponds to a good structural match and synergy between biominerals and substrates will promote an ordered, compact, and tough complex biomineral structure, and occur only at low supersaturations, whereas at high supersaturations the heterogeneous nucleation associated with a poor structural match and synergy between biominerals and substrates will become dominant due to supersaturation-driven interfacial structural mismatch. The latter normally results in a disordered and porous structure. A phenomenon, so-called microgravity-driven homogeneous nucleation, was also examined. It turns out that microgravity will suppress convection and consequently promote homogeneous-like nucleation during biomineralization. This could be responsible for microgravity-induced osteoporousis.

Calcium Carbonate↗

An alternative interpretation of nanobacteria-induced biomineralization.

The reported isolation of nanobacteria from human kidney stones raises the intriguing possibility that these microorganisms are etiological agents of pathological extraskeletal calcification [Kajander, E. O. & Ciftçioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274-8279]. Nanobacteria were previously isolated from FBS after prolonged incubation in DMEM. These bacteria initiated biomineralization of the culture medium and were identified in calcified particles and biofilms by nucleic acid stains, 16S rDNA sequencing, electron microscopy, and the demonstration of a transferable biomineralization activity. We have now identified putative nanobacteria, not only from FBS, but also from human saliva and dental plaque after the incubation of 0.45-microm membrane-filtered samples in DMEM. Although biomineralization in our "cultures" was transferable to fresh DMEM, molecular examination of decalcified biofilms failed to detect nucleic acid or protein that would be expected from growth of a living entity. In addition, biomineralization was not inhibited by sodium azide. Furthermore, the 16S rDNA sequences previously ascribed to Nanobacterium sanguineum and Nanobacterium sp. were found to be indistinguishable from those of an environmental microorganism, Phyllobacterium mysinacearum, that has been previously detected as a contaminant in PCR. Thus, these data do not provide plausible support for the existence of a previously undiscovered bacterial genus. Instead, we provide evidence that biomineralization previously attributed to nanobacteria may be initiated by nonliving macromolecules and transferred on "subculture" by self-propagating microcrystalline apatite.

Bacteria↗

Biomineralization: new directions in crystal science.

Effective protocols for controlling crystal structure, size, and morphology attract considerable interest given the requirement for particles of modal size and shape in many areas of materials fabrication and the importance of crystallochemical selectivity in determining the exploitable properties of inorganic solids. For this reason biomineralization merits particular attention since many biominerals are deposited in a highly controlled manner to produce crystals which are uniformly sized and crystallographically unique. Studies of biominerals have revealed that while a complex array of strategies have evolved for regulating their formation, one feature is common to the biological paradigm; interactions between organized biopolymeric assemblies and the nascent inorganic solids play a pivotal role in controlling the crystallization process. In order to gain a better understanding of the molecular interactions which take place at organic-inorganic interface and address the fundamental chemical problems of biomineralization, a crystal chemical approach has been adopted. Organized organic assemblies (phospholipid vesicles, Langmuir monolayers, polypetide templates) of precise molecular design (head group identity, packing conformation, peptide sequence, etc.) were assayed for their effectiveness in controlling the nucleation and growth of inorganic solids. This work has established that through systematic changes in the nature of the organic matrix the size, crystallographic orientation, and growth of the mineral phase can be controlled. Critical to this process was the translation of specific molecular information at the organic-inorganic interface: epitaxial alignment, stereochemical complementarity, and electrostatic interactions were an essential feature of this recognition event.

Animals↗

Calcium biomineralization in the radular teeth of the chiton, Acanthopleura hirtosa.

A method has been devised for isolating the calcium biomineral from the iron biominerals and organic components present in the major lateral teeth of the chiton Acanthopleura hirtosa. Fourier-transform infrared spectroscopy of the calcium biomineral indicated that it was an apatite material containing carbonate and fluoride ions. Carbonate was not found to be present as a separate phase. The apatite was further separated into low and high density fractions, both of which showed crystallinity intermediate between that of bovine tibia cortical bone and human tooth enamel, as indicated by powder X-ray diffraction analysis. The calcified region of the major lateral teeth was also studied in situ using transmission electron microscopy and electron diffraction analysis, revealing a close spatial relationship between the mineral apatite phase and underlying organic matrix. It is suggested that the architectural arrangement of apatite biomineral and fibrous organic constituents imparts specialized mechanical properties to the tooth making it ideally suited for the task of obtaining food from hard surfaces.

Animals↗

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis.

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a = 9.382 A and c = 6.883 A, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.

Animals↗

Biomineralization and adaptive plasticity of the temporomandibular joint in myostatin knockout mice.

Mice lacking myostatin (GDF-8), a negative regulator of skeletal muscle growth, show a significant increase in muscle mass versus normal mice. We compared wild-type and myostatin deficient mice to assess the postnatal effect of elevated masticatory loads due to increased jaw-adductor muscle activity and greater bite forces on mandibular condyle morphology. Microcomputed tomography (microCT) was used to provide details of internal condylar morphology and quantify bone density in three condylar regions. Biomineralization levels, as well as external mandibular dimensions, were used to characterize within-slice, within-joint, within-group and between-group variation. Dimensions of the mandible and mandibular condyle were similar between the myostatin knockout and normal mice. Knockout mice exhibited significantly more biomineralization on the outer surface of the condylar subchondral bone and along the condylar neck, most notably on the buccal side of the condylar neck. The buccal side of the inner aspect of the condyle was significantly less biomineralized in knockout mice, both for the pooled data and for the posterior and anterior condylar slices. Whilst normal mice had symmetric subchondral bone surfaces, those of knockout mice were asymmetric, with a lower, less convex surface on the buccal side versus the lingual side. This appears related to the ontogenetic effects of increased masticatory stress in the mandibles of knockout mice as compared to normal mice. Significant differences in biomineralization between normal and myostatin knockout mice, coupled with the lack of significant differences in certain external dimensions, underscores a need for information on the external and internal morphology of mineralized tissues vis-à-vis altered or excessive mechanical loads.

Adaptation, Physiological↗

Developmental biology meets materials science: Morphogenesis of biomineralized structures.

Biomineralization is the process by which metazoa form hard minerals for support, defense, and feeding. The minerals so formed, e.g., teeth, bones, shells, carapaces, and spicules, are of considerable interest to chemists and materials scientists. The cell biology underlying biomineralization is not well understood. The study of the formation of mineralized structures in developing organisms offers opportunities for understanding some intriguing aspects of cell and developmental biology. Five examples of biomineralization are presented: (1) the formation of siliceous spicules and frustules in sponges and diatoms, respectively; (2) the structure of skeletal spicules composed of amorphous calcium carbonate in some tunicates; (3) the secretion of the prism and nacre of some molluscan shells; (4) the development of skeletal spicules of sea urchin embryos; and (5) the formation of enamel of vertebrate teeth. Some speculations on the cellular and molecular mechanisms that support biomineralization, and their evolutionary origins, are discussed.

Animals↗

Microbial fuel cell using anaerobic respiration as an anodic reaction and biomineralized manganese as a cathodic reactant.

We have operated a microbial fuel cell in which glucose was oxidized by Klebsiella pneumoniae in the anodic compartment, and biomineralized manganese oxides, deposited by Leptothrix discophora, were electrochemically reduced in the cathodic compartment. In the anodic compartment, to facilitate the electron transfer from glucose to the graphite electrode, we added a redox mediator, 2-hydroxy-1,4-naphthoquinone. We did not add any redox mediator to the cathodic compartment because the biomineralized manganese oxides were deposited on the surface of a graphite electrode and were reduced directly by electrons from the electrode. We have demonstrated that biomineralized manganese oxides are superiorto oxygen when used as cathodic reactants in microbial fuel cells. The current density delivered by using biomineralized manganese oxides as the cathodic reactant was almost 2 orders of magnitude higher than that delivered using oxygen. Several fuel cells were operated for 500 h, reaching anodic potentials of -441.5 +/- 31 mVscE and cathodic potentials of +384.5 +/- 64 mVscE. When the electrodes were connected by a 50 Ohms resistor, the fuel cell delivered the peak power density of 126.7 +/- 31.5 mW/m2.

Bioelectric Energy Sources↗

Altering biomineralization by protein design.

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.

Ameloblasts↗

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

Calcification, Physiologic↗

[Biomineralization at hot springs and mineral springs, and their significance in relation to the Earth's history].

Recently, there is strong interest on microbe-mineral interactions. This is related also to recent expanded knowledges on extremely severe environments in which microbes live. Interaction between microbes and minerals contains biomineralization processes. Varieties of biomineralization products are found not only in various geologic materials and processes in the earth's history but also in present surface environments. Some hot springs represent such environments similar to those of unique and extremely severe environments for life. In this short review, the author briefly shows some examples of biomineralizations at some hot springs and mineral springs, Japan. In such environments, iron ore was formed and some varieties of growing stromatolites were found. The varieties of stromatolite are siliceous, calcic and manganese types. Cyanobacteria and the other bacteria are related to form the stromatolite structure. In the Gunma iron ore, sedimentary iron ores were mineralogically described in order to evaluate the role of microorganisms and plants in ore formation. The iron ore is composed of nanocrystalline goethite. Algal fossils are clearly preserved in some ores. Various products of biomineralization are found in the present pH 2-3, Fe2(+)- and SO4(2-)-rich streams. Bacterial precipitation had variations from amorphous Fe-P-(S) precipitates near the outlet of mineral spring, to Fe-P-S precipitates and to Fe-S-(P) precipitates. Mosses and green algae are also collecting Fe precipitates in and around the living and dead cells. The Gunma Iron Ore can be said as Biologically Induced Iron Ore. At Onikobe and Akakura hot springs, growing stromatolites of siliceous and calcareous types, were found, respectively. At Onikobe, The stromatolites grow especially near the geyser. Cyanobacterial filaments in stromatolite were well preserved in the siliceous and calcic stromatolites. The filaments oriented in two directions which form the layered structures were found. At Yunokoya hot spring, black and brittle stromatolitic structures which were composed of amorphous Mn minerals are growing. The form of these structures are hemispherical. Many bacteria that were coated with amorphous Mn minerals were found on these structures. Furthermore, Precambrian (Proterozoic : Wittenoom-Chichester region, western Australia) manganese stromatolite was briefly shown in comparison. The black stromatolite has been clarified to be composed of todorokite. Small spotty and donuts-like shaped todorokite aggregates which are very similar to biologically induced Mn-precipitates were found in massive dolomite layers.

Australia↗