Extracellular Biomineralization Research Articles

Uranium-tolerant soil bacteria protect Arabidopsis thaliana seedling growth from uranium toxicity

Uranium (U) is a naturally occurring radioelement that is chemically toxic to all living organisms. We investigated the effect of U on Arabidopsis thaliana seed germination and the early stages of seedling development. We showed that U had very little effect on Arabidopsis seed germination, with germination rates above 90 % at 500 µM uranyl nitrate. However, U had a drastic effect on the Arabidopsis seedling development and the root was the most U-sensitive organ, with severe growth inhibition (>75 %) starting at 20 µM uranyl nitrate. We showed that the two soil bacterial strains Microbacterium sp. ViU2A and Stenotrophomonas bentonitica BII-R7, which are able to tolerate high concentrations of U, strongly reduced the toxic effects of U on seedling development. Thus, in the presence of both strains, root growth inhibition was prevented up to 100 µM uranyl nitrate. This protection was specific to U-tolerant soil bacteria, as Escherichia coli was unable to protect seedlings. The analysis of U distribution between Arabidopsis seedlings and soil bacteria showed that the protective effect of bacteria was due to their ability to sequester U, either by biosorption at the cell surface and/or by intracellular or extracellular biomineralization. Also, the ability of both soil bacterial strains to prevent U toxicity was much less efficient when dead bacteria were used, suggesting the involvement of metabolism-dependent biological processes in seedling protection. Accordingly, we showed that the Uranium-induced protein A (UipA) contributes to the protection of Arabidopsis seedlings by Microbacterium sp. ViU2A. This study suggests that U-tolerant soil bacteria may be useful for phytostabilization strategies of U-contaminated soils and for limiting the entry of this toxic element into crops.

The Development of Limestone Weathering Rind in a Proglacial Environment of the Hallstätter Glacier

The contemporary retreat of glaciers results in the development of glacial forelands which constitute excellent areas for studies on the initial stages of weathering. This research concentrates on weathering rinds developed on glacially abraded Dachstein limestone surfaces (Eastern Alps) liberated from glacial ice since LIA (mid-19th century). We performed measurements of rock surface micro-roughness and strength (Schmidt hammer), spectral reflectance, and petrographic and microbiological analyses in SEM. There was a time-dependent increase in micro-roughness, decrease in rock strength and decrease in spectral reflectivity, but only within visual light. Rock surfaces on older sites reflected infrared radiation significantly better than younger ones. SEM analysis revealed that angular micro-structures inherited from glacial abrasion dominated in younger sites, eventually giving way to more rounded micro-structures. The structure of rock (micrite versus spar, phlogopite veinlets, etc.) plays a significant role. On older sites, microorganisms were detected within the studied weathering rinds, which probably results in better infrared reflectivity. We advocate the potential role of microorganisms (mainly heterotrophic) in the dissolution of limestone, and the role of cyanobacteria in the formation of the secondary porous limestone layer (extracellular biomineralization).

Extracellular biomineralization of uranium and its toxicity alleviation to Bacillus thuringiensis X-27

Uranium biomineralization can slow uranium migration in the environment and thus prevent it from further contaminating the surroundings. Investigations into the uranium species, pH, inorganic phosphate (Pi) concentration, and microbial viability during biomineralization by microorganisms are crucial for understanding the mineralization mechanism. In this study, Bacillus thuringiensis X-27 was isolated from soil contaminated with uranium and was used to investigate the formation process of uranium biominerals induced by X-27. The results showed that as biomineralization proceeded, amorphous uranium-containing deposits were generated and transformed into crystalline minerals outside cells, increasing the overall concentration of uramphite. This is a cumulative rather than abrupt process. Notably, B. thuringiensis X-27 precipitated uranium outside the cell surface within 0.5 h, while the release of Pi into the extracellular environment and the change of pH to alkalescence further promoted the formation of uramphite. In addition, cell viability determination showed that the U(VI) biomineralization induced by B. thuringiensis X-27 was instrumental in alleviating the toxicity of U(VI) to cells. This work offers insight into the mechanism of U(VI) phosphate biomineralization and is a reference for bioremediation-related studies.

Calcium ion removal at different sodium chloride concentrations by free and immobilized halophilic bacteria

Much attention has been paid to Ca2+ ion removal by biomineralization due to the dangers of Ca2+ on industrial processes and human health. However, Ca2+ removal from hypersaline water by biomineralization is quite difficult due to there being few halophilic bacteria tolerating higher salinities. In this study, free and immobilized Virgibacillus massiliensis C halophilic bacteria exhibiting carbonic anhydrase activity were used to remove Ca2+ ions from water at different NaCl concentrations. With increasing NaCl concentrations (10, 50, 100, 150 and 200 g/L), Ca2+ ion concentrations in the presence of free bacteria and in two groups of immobilized bacteria for a period of 6 days sharply decreased from 1200 mg/L to 219–562 mg/L, 71–214 mg/L and 21–159 mg/L, respectively; Ca2+ precipitation ratios were 55%-81%, 82%-94% and 87%-98%, respectively. The humic acid-like substances, protein, DNA and polysaccharide, released by the bacteria, promoted the Ca2+ ion removal. The immobilized bacteria were able to be recycled and precultured, which would save industry costs and increase Ca2+ ion removal efficiency. Biological processes for Ca2+ ion removal include cell surface, intracellular and extracellular biomineralization. The biogenesis of calcium carbonate was proved by SEM-EDS, FTIR, XPS and stable carbon isotope values. This study provides insights into the effective removal of Ca2+ ions by biomineralization in hypersaline water.

Extracellular, Surface, and Intracellular Biomineralization of Bacillus subtilis Daniel-1 Bacteria

Even though biomineralization induced by bacteria has been studied for many years, some unsolved problems are still present in the biomineralization, especially the intracellular biomineralization, which should be further explored. In this study, the Bacillus subtilis Daniel-1 strain was identified by 16S rDNA homology, used to induce calcium carbonate biomineralization at various Mg/Ca molar ratios (0, 2, 4, 6, and 8). The results showed that pH still increased at the decline stage in the bacterial growth process, and could increase to about 8.69, resulting from the influence of carbonic anhydrase activity and ammonia. X-ray diffraction (XRD) results showed that calcite, Mg-rich calcite, and aragonite minerals were induced in the extracellular biomineralization process. The diversified morphologies and the presence of fatty acids, protein, and other organic substances in calcium carbonate analyzed by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) proved the biogenesis of these minerals. The stable carbon isotope values (–19.37‰ to −19.79‰) and oxygen isotope values (–6.37‰ to −8.91‰) presented in this study were relatively negative, also confirmed the biogenesis of the minerals. Magnesium carbonate contents in Mg-rich calcite increased from 8.79% to 25.71% with increasing Mg/Ca ratios. Aragonite could be grown not only in cell surface but also inside the cell by analyzing ultrathin slices of bacteria with HRTEM-EDS-SAED. Aragonite in cell surface was elongated whereas intracellular aragonite was granular. To the best of our knowledge, the intracellular biotic crystalline aragonite has rarely been reported. This study is helpful to further understand the mechanism of carbonate mineral biomineralization, and may also provide useful evidence for further studies on intracellular crystal structures.

Benefits at the nanoscale: a review of nanoparticle‐enabled processes favouring microbial growth and functionality

Nanoparticles are ubiquitous and co-occur with microbial life in every environment on Earth. Interactions between microbes and nanoparticles impact the biogeochemical cycles via accelerating various reaction rates and enabling biological processes at the smallest scales. Distinct from microbe-mineral interactions at large, microbe-nanoparticle interactions may involve higher levels of active recognition and utilization of the reactive, changeable, and thereby 'moldable' nano-sized inorganic phases by microbes, which has been given minimal attention in previous reviews. Here we have compiled the various cases of microbe-nanoparticle interactions with clear and potential benefits to the microbial cells and communities. Specifically, we discussed (i) the high bioavailabilities of nanoparticles due to increased specific surface areas and size-dependent solubility, with a focus on environmentally-relevant iron(III) (oxyhydr)oxides and pyrite, (ii) microbial utilization of nanoparticles as 'nano-tools' for electron transfer, chemotaxis, and storage units, and (iii) speculated benefits of precipitating 'moldable' nanoparticles in extracellular biomineralization. We further discussed emergent questions concerning cellular level responses to nanoparticle-associated cues, and the factors that affect nanoparticles' bioavailabilities beyond size-dependent effects. We end the review by proposing a framework towards more quantitative approaches and by highlighting promising techniques to guide future research in this exciting field.

ToF-SIMS 3D imaging unveils important insights on the cellular microenvironment during biomineralization of gold nanostructures

The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra- and extracellular biomineralization. At low Au concentrations (≤1 mM HAuCl4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (≥2 mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology.

Intracellular and Extracellular Biomineralization Induced by Klebsiella pneumoniae LH1 Isolated from Dolomites

The interaction between bacteria and minerals is very complicated and has been intensively studied in the laboratory and the field in the last few decades, but the processes and mechanisms of biomineralization and mineral precipitation are still not fully understood and need to be explored further. In the present work, biomineralization experiments were undertaken using Klebsiella pneumoniae LH1, collected from a natural surface environment in an area of outcrops of Cambrian dolomite, in a culture medium with various Mg/Ca molar ratios (0, 3, 6 and 12). The mineral precipitates obtained were analyzed by X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive spectrometer (EDS), Fourier transform infrared spectrometer (FTIR), laser scanning confocal microscopy (LSCM) and X-ray photoelectron spectroscopy (XPS). Cells were analyzed with a scanning transmission electron microscope (STEM), high resolution transmission electron microscope (HRTEM) and selected area electron diffraction (SAED). The composition of amino acids in extracellular polymeric substances (EPS) was also determined. In the experiments it was found that the production of ammonia and the presence of carbonate anhydrase promoted the increase of the medium pH and that minerals are nucleated on the EPS, which consist chiefly of amino acids and negatively-charged organic functional groups. With increasing Mg/Ca ratios, the mineral phases changed, including calcite (100%) at Mg/Ca molar ratio of 0, monohydrocalcite (36.05%) + dypingite (63.95%) at Mg/Ca molar ratio of 3, monohydrocalcite (29.72%) + dypingite (15.48%) + nesquehonite (54.80%) at Mg/Ca molar ratio of 6, and monohydrocalcite (14.2%) + dypingite (1.0%) + nesquehonite (84.80%) at Mg/Ca molar ratio of 12. Some intracellular amorphous calcium- and magnesium-rich inclusions were also detected in K. pneumoniae LH1, suggesting intracellular biomineralization accompanying the extracellular mineral precipitation. This study provides further understanding of the biomineralization processes of microorganisms.

Biomineralization of Monohydrocalcite Induced by the Halophile Halomonas Smyrnensis WMS-3

The halophilic bacterium Halomonas smyrnensis from a modern salt lake used in experiments to induce biomineralization has resulted in the precipitation of monohydrocalcite and other carbonate minerals. In this study, a Halomonas smyrnensis WMS-3 (GenBank:MH425323) strain was identified based on 16S rDNA homology comparison, and then cultured in mediums with 3% NaCl concentration to induce monohydrocalcite at different Mg/Ca molar ratios of 0, 2, 5, 7, and 9. The growth curve of WMS-3 bacteria, pH values, NH4+ concentration, HCO3− and CO32− concentration, carbonic anhydrase (CA) activity, and the changes in Ca2+ and Mg2+ ion concentration were determined to further explore the extracellular biomineralization mechanism. Moreover, the nucleation mechanism of monohydrocalcite on extracellular polymeric substances (EPS) was analyzed through studying ultrathin slices of the WMS-3 strain by High resolution transmission electron microscopy (HRTEM), Selected area election diffraction (SAED), Scanning transmission electron microscopy (STEM), and elemental mapping, besides this, amino acids in the EPS were also analyzed. The results show that pH increased to about 9.0 under the influence of ammonia and CA activity. The precipitation ratio (%, the ratio of the mass/volume concentration) of the Ca2+ ion was 64.32%, 62.20%, 60.22%, 59.57%, and 54.42% at Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively, on the 21st day of the experiments, and 6.69%, 7.10%, 7.74%, 8.09% for the Mg2+ ion concentration at Mg/Ca molar ratios 2, 5, 7, and 9, respectively. The obtained minerals were calcite, Mg-rich calcite, aragonite, and hydromagnesite, in addition to the monohydrocalcite, as identified by X-ray diffraction (XRD) analyses. Monohydrocalcite had higher crystallinity when the Mg/Ca ratio increased from 7 to 9; thus, the stability of monohydrocalcite increased, also proven by the thermogravimetry (TG), derivative thermogravimetry (DTG) and differential scanning calorimetry (DSC) analyses. The C=O and C–O–C organic functional groups present in/on the minerals analyzed by Fourier transform infrared spectroscopy (FTIR), the various morphologies and the existence of P and S determined by scanning electron microscope-energy dispersive spectrometer (SEM-EDS), the relatively more negative stable carbon isotope values (−16.91‰ to −17.91‰) analyzed by a carbon isotope laser spectrometer, plus the typical surface chemistry by XPS, all support the biogenesis of these mineral precipitates. Moreover, Ca2+ ions were able to enter the bacterial cell to induce intracellular biomineralization. This study is useful to understand the mechanism of biomineralization further and may provide theoretical reference concerning the formation of monohydrocalcite in nature.

Extracellular Biomineralization of Gold by Delftia tsuruhatensis GX-3 Isolated from a Heavy Metal Contaminated Paddy Soil

The use of microorganisms in geochemical gold recycling was considered recently. To explore the feasibility of gold biomineralization in this study, bacteria GX-3 were isolated from a heavy metal contaminated paddy soil in Guangxi province, China. This sulfur-reducing bacteria, Delftia tsuruhatensis, can survive in toxic Au3+ solution. Alkaline conditions (pH 8.0) and 35 °C were optimal for GX-3 cultivation. Nanoparticle formation was promoted by the coexistence of Fe3+. Field-emission scanning electron microscopy and transmission electron microscopy (TEM) images of GX-3 showed the presence of spherical particles, ranging from 50 to 100 and 200 to 500 nm in diameter, respectively, that stuck together and distributed around the surface of the bacteria as well as outside the cell. These nanoparticles were identified as gold according to the TEM–energy dispersive X-ray spectroscopy analysis. Here, bacterial extracellular solute-binding protein and/or porin should account for the gold nanoparticle formation and the detoxification in aqueous system as it transports electrons to Au3+ for nanoparticle precipitation. The ubiquitous nature of bacteria in near-surface environments and the rapid precipitation of gold by the sulfur-reducing bacterium suggest that these bacteria could be responsible for the wide bioaccumulation of gold in natural systems.

Concurrent biomineralization of silver ions into Ag0 and AgxO by Leptolyngbya strain JSC-1 and the establishment of its axenic culture

Ionic silver is a potential hazard to aquatic life forms because of the increasing usage of silver based materials. The need for developing a sustainable and ecofriendly process to minimize the toxic effects of the free ions burden is now a scientific consensus. Therefore, we report the latest results in cyanobacterium Leptolyngbya JSC-1 investigating the tolerance towards toxic doses of silver, its extracellular biomineralization and silver nano-deposits formation inside the cells, and speculate about potential environmental impacts. In this study, scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDS) analysis reveal the extracellular biomineralization of soluble silver (1–100 μM) into corresponding nanoparticles (50–100 nm in diameter) by JSC-1, while X-ray photoelectron spectroscopy (XPS) examination divulged the presence of both Ag+ and Ag0 in extracellularly biomineralized silver, depicting a mixture of both AgxO and elemental Ag. The scanning transmission electron microscopy (STEM), EDS and elemental mapping visualized the formation of intracellular silver nanoparticles. Moreover, this feature of silver tolerance in JSC-1 was further exploited and a novel protocol was developed for isolation and maintenance of axenic culture of this filamentous cyanobacterium. Consequently, this capability of silver biomineralization by JSC-1, both extra- and intra-cellularly might be useful for modeling the Ag resistance mechanism in cyanobacteria and also might be a sustainable alternative for heavy metals bioremediation in aquatic environments.

Biomineralized CdS Quantum Dot Nanocrystals: Optimizing Synthesis Conditions and Improving Functional Properties by Surface Modification

An engineered strain of Stenotrophomonas maltophilia (SMCD1) is capable of the direct extracellular biomineralization of CdS quantum dot nanocrystals from buffered aqueous solution of cadmium acetate and l-cysteine without the addition of a chemically reactive precursor. Nanocrystal synthesis is strongly influenced by both the l-cysteine/cadmium acetate ratio and pH of the solution. The observed trends are consistent with l-cysteine acting as both a sulfur source and nanocrystal capping agent. Enzymatic turnover of l-cysteine by a putative cystathionine γ-lyase forms reactive sulfur in solution, removing the requirement for addition of reactive sodium sulfide typical of most other biomineralization approaches. The utility of the biomineralized quantum dots is demonstrated by phase transfer from the aqueous to the organic phase and subsequent incorporation into a quantum dot sensitized solar cell and chemical growth of a ZnS shell onto the biomineralized CdS core.

The Characterization of Intracellular and Extracellular Biomineralization Induced by Synechocystis sp. PCC6803 Cultured under Low Mg/Ca Ratios Conditions

ABSTRACTIn order to simulate the precipitation process of microbial limestone at the offshore of the ancient ocean, different calcites induced by Synechocystis sp. PCC6803 in culture media with low Mg/Ca ratios (0.01 M Ca2+, Mg/Ca = 0, 0.2, 0.4, 0.6) were investigated, and the characteristics of intracellular and extracellular biomineralization were described. Carbonic anhydrase activity of Synechocystis sp. PCC6803 in different culture medium was further detected. The ultrathin slices of Synechocystis sp. PCC6803 cells were analyzed by transmission electron microscope (TEM) and selected area electron diffraction (SAED). Then the precipitates were analyzed by polarizing microscope, scanning electron microscope (SEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The results showed that the biomineralization precipitates of Synechocystis sp. PCC6803 under low Mg/Ca ratios were mainly calcites with different morphologies. The CA accelerated the pivotal rate limiting step of the calcite precipitation. It was also found that the morphology, microstructure, particle size, preferred orientation, crystallinity and cell volume of calcites changed gradually with the increasing Mg2+ concentrations. What is more important, it was found that Synechocystis sp. PCC6803 had the ability of intracellular biomineralization without crystal structure. The intracellular biomineralization product could be divided into two types. This study can provide some useful information for further understanding the characteristics and mechanisms of biomineralization and even the diagenetic environment research of microbial limestone.

Efficient trafficking of acidic proteins out of the endoplasmic reticulum involves a conserved amino terminal IleProVal (IPV)-like tripeptide motif

Most of the proposed extracellular biomineralization processes include the secretion of proteins that interact with mineral ions and/or mineral surfaces. Typically these proteins are acidic or have acidic domains that interact with multivalent cations in the extracellular environment. We propose that most acidic, Ca2+-binding proteins challenge the cell’s mechanisms for trafficking through the endoplasmic reticulum (ER) lumen due to lumenal mM calcium that cause them to form large aggregates. We have recently shown that >95% of the DSPP mutations that cause non-syndromic genetic dentin diseases start their dominant negative affects by failing to rapidly exit the ER likely by forming complexes that cannot be normally trafficked to the Golgi. The complexes of mutant DSPP then capture more (severe disease) or less (mild disease) of the DSPP translated from the normal allele. After searching genomic databases as well as the published literature, we found the IleProVal (IPV)-like motif at the predicted amino terminus of many acidic proteins made in the mineralizing as well as non-mineralizing tissues of many species including vertebrates, echinoderms, mollusks, and yeast. While we often focused on acidic proteins reported associated with mineralizing structures, proteins associated with hormones and their storage/secretion, digestion, blood functions, as well as milk and other secreted fluids started with variations of the motif. Our hypothesis is that the IPV-like motif interacts with a highly conserved cargo receptor in the ER that efficiently traffics the acidic proteins out of the organelle before they can form harmful aggregates in the Ca2+-rich lumen.

Proteomics of CaCO3biomineral-associated proteins: How to properly address their analysis

In a recent editorial (Proc. Natl. Acad. Sci., 2013 110, E2144-E2146) and elsewhere, questions have been raised regarding the experimental practices in relation to the proteomic analysis of organic matrices associated to the biomineralized CaCO3 skeletons of metazoans such as molluscan shells and coral skeletons. Indeed, although the use of new high sensitivity MS technology potentially allows to identify a greater number of proteins, it is also equally (or even more) sensitive to contamination of residual proteins from soft tissues, which are in close contact with the biomineral. Based on our own past and present experimental know-how-observations that are reproducible and coherent with the current understanding of extracellular biomineralization processes-we are convinced that a careful and appropriate cleaning of biominerals prior to any analysis is crucial for accurate proteomic investigations and subsequent pertinent interpretation of the results. Our goal is to alert the scientific community about the associated bias that definitely should be avoided, and to provide critical recommendations on sample preparation and experimental design, in order to better take advantage of the aptness of proteomic approaches aiming at improving our understanding of the molecular mechanisms in biomineralization.

Direct Observation of Phenylalanine Orientations in Statherin Bound to Hydroxyapatite Surfaces

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

Biological Synthesis of Gold Nanoparticles Using the Aqueous Extract of the Brown Algae Laminaria Japonica

In this study, the extracellular biosynthesis of self-assembled gold nanoparticles (GNPs) was accomplished using the aqueous extract of the brown algae Laminaria japonica. The optical, physical, and chemical properties of the GNPs were investigated to identify their average shape and size, crystal nature, and surface chemistry, via UV-visible spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The sizes of the spherical self-assembled cores of the GNPs ranged from 15 to 20 nm. The XRD patterns showed a (111) preferential orientation and the crystalline nature of the GNPs. The results of the FTIR analysis suggested that the peptides and/or proteins carried out the dual function of effective AuIII reduction and successful capping of the GNPs. Thus, the biomolecular synthesis, stabilization, and self-assembly of the GNPs by L. japonica extract can be an example of green chemistry and extracellular biomineralization.

Characterization of Fe-S minerals influenced by buried ancient woods in the intertidal zone, East China Sea

14 C dating was discovered in the intertidal zone, East China Sea. Samples affected by ancient woods, including fresh coast bedrock, weathering bedrock, seepage water from coast, seepage water from ancient wood layer, intertidal seawater, fresh water, beach mud, ancient wood barks and ancient peat, were collected for geochemical analysis. The beach mud and the bacteriogenic iron oxides (BIOS) in coastal seepage water were analyzed by min- eralogical and high-resolution transmission electron microscopy (HRTEM)-selected area electron dif- fraction (SAED) analysis. Inorganic sulfur compositions and δ 34 S of the ancient peat and the beach mud were determined. The results showed that Fe, Mn, S (SO4− ) were enriched in the intertidal area at dif- ferent levels, very likely caused by fermentation of ancient woods. The presence of abundant iron- oxidizing bacteria (FeOB) and sulfate-reducing bacteria (SRB) in this intertidal zone was confirmed by HRTEM-SAED observation, and these bacteria were involved in Fe-S cycle to induce extracellular biomineralization. The negative δ 34 SV-CDT (−2.9‰) likely indicated the biogenic origin of iron-sulfide minerals in the beach mud at high sulfate reduction rate (SRR). These findings are helpful for under- standing the biogeochemical Fe-S cycle and biomineralization process at high organic matter deposition rate and high SRR in the intertidal zone, estuary, or near shoreline.

Confocal Laser Raman Microspectroscopy of Biomineralization Foci in UMR 106 Osteoblastic Cultures Reveals Temporally Synchronized Protein Changes Preceding and Accompanying Mineral Crystal Deposition

Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of beta-glycerol phosphate to cells at 64 h after plating. BMF are identified by their enrichment with an 85-kDa glycoprotein reactive with Maackia amurensis lectin. Laser Raman microspectroscopic scans were made on individual BMF at times preceding (64-76 h) and following the appearance of mineral crystals (76-88 h). The range of variation between spectra for different BMF in the same culture was rather small. In contrast, significant differences were observed for spectral bands at 957-960, 1004, and 1660 cm(-1) when normalized BMF spectra at different times were compared. Protein-dependent spectral bands at 1004 and 1660 cm(-1) increased and then decreased preceding the detection of hydroxyapatite crystals via the phosphate stretching peak at 959-960 cm(-1). When sodium phosphate was substituted for beta-glycerol phosphate, mineralization occurred 3-6 h earlier. Irrespective of phosphate source, the Raman full peak width at half-maximum ratio for 88 h cultures was similar to that for 10-day-old marrow ablation primary bone. However, if mineralization was blocked with serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 64-88-h BMF spectra remained largely invariant. In summary, Raman spectral data demonstrate for the first time that formation of hydroxyapatite crystals within individual BMF is a multistep process. Second, changes in protein-derived signals at 1004 and 1660 cm(-1) reflect events within BMFs that precede or accompany mineral crystal production because they are blocked by mineralization inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Finally, the low extent of spectral variability detected among different BMF at the same time point indicates that mineralization of individual BMF within a culture is synchronized.

Identification and Structural Characterization of an Unusual RING-Like Sequence within an Extracellular Biomineralization Protein, AP7

The RING or Really Interesting New Gene represents a family of eukaryotic sequences that bind Zn (II) ions and participate in intracellular processes involving protein-protein interaction. Although found in over 400 different proteins, very little is known regarding the structure-function properties of these domains because of the aggregation problems associated with RING sequences. To augment this data set, we report an unusual 36 AA C-terminal sequence of an extracellular matrix mollusk shell protein, AP7, that exhibits partial homology to the RING family. This Cys, His-containing sequence, termed AP7C, binds Zn (II) and other multivalent ions, but does not utilize a tetracoordinate complexation scheme for binding such as that found in Zn (II) finger polypeptides. Moreover, unlike Zn (II) finger and RING domains, this 36 AA can fold into a relatively stable structure in the absence of Zn (II). This folded structure consists of three short helical segments (A, B, and C), with segments A and B separated by a 4 AA type I beta-turn region and segments B and C separated by a 7 AA loop-like region. Interestingly, the putative RING-like region, -RRPFHECALCYSI-, experiences slow conformational exchange between two structural states in solution, most likely in response to imido ring interconversion at P8 and P21. Poisson-Boltzmann solvation calculations reveal that the AP7C molecular surface possesses a cationic region near its N-terminus, which lies adjacent to the 30 AA mineral modification domain in the AP7 protein. Given that the AP7C sequence does not influence mineralization, it is probable that this cationic pseudo-RING region is utilized by the AP7 protein for other tasks such as protein-protein interaction within the mollusk shell matrix.