Induced Mineralization of Hydroxyapatite in Escherichia coli Biofilms and the Potential Role of Bacterial Alkaline Phosphatase

Biofilms appear when bacteria colonize a surface and synthesize and assemble extracellular matrix components. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphate. While calcified biofilms induce diseases like periodontitis in physiological environments, they also inspire the engineering of living composites. Understanding mineralization mechanisms in biofilms will thus provide key knowledge for either inhibiting or promoting mineralization in these research fields. In this work, we study the mineralization of Escherichia coli biofilms using the strain E. coli K-12 W3110, known to produce an amyloid-based fibrous matrix. We first identify the mineralization conditions of biofilms grown on nutritive agar substrates supplemented with calcium ions and β-glycerophosphate. We then localize the mineral phase at different scales using light and scanning electron microscopy in wet conditions as well as X-ray microtomography. Wide-angle X-ray scattering enables us to further identify the mineral as being hydroxyapatite. Considering the major role played by the enzyme alkaline phosphatase (ALP) in calcium phosphate precipitation in mammalian bone tissue, we further test if periplasmic ALP expressed from the phoA gene in E. coli is involved in biofilm mineralization. We show that E. coli biofilms grown on mineralizing medium supplemented with an ALP inhibitor undergo less and delayed mineralization and that purified ALP deposited on mineralizing medium is sufficient to induce mineralization. These results suggest that also bacterial ALP, expressed in E. coli biofilms, can promote mineralization. Overall, knowledge about hydroxyapatite mineralization in E. coli biofilms will benefit the development of strategies against diseases involving calcified biofilms as well as the engineering of biofilm-based living composites

Induced Mineralization of Hydroxyapatite in Escherichia coli Biofilms and the Potential Role of Bacterial Alkaline Phosphatase

Biofilms appear when bacteria colonize a surface and synthesize and assemble extracellular matrix components. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphate. While calcified biofilms induce diseases like periodontitis in physiological environments, they also inspire the engineering of living composites. Understanding mineralization mechanisms in biofilms will thus provide key knowledge for either inhibiting or promoting mineralization in these research fields. In this work, we study the mineralization of Escherichia coli biofilms using the strain E. coli K-12 W3110, known to produce an amyloid-based fibrous matrix. We first identify the mineralization conditions of biofilms grown on nutritive agar substrates supplemented with calcium ions and β-glycerophosphate. We then localize the mineral phase at different scales using light and scanning electron microscopy in wet conditions as well as X-ray microtomography. Wide-angle X-ray scattering enables us to further identify the mineral as being hydroxyapatite. Considering the major role played by the enzyme alkaline phosphatase (ALP) in calcium phosphate precipitation in mammalian bone tissue, we further test if periplasmic ALP expressed from the phoA gene in E. coli is involved in biofilm mineralization. We show that E. coli biofilms grown on mineralizing medium supplemented with an ALP inhibitor undergo less and delayed mineralization and that purified ALP deposited on mineralizing medium is sufficient to induce mineralization. These results suggest that also bacterial ALP, expressed in E. coli biofilms, can promote mineralization. Overall, knowledge about hydroxyapatite mineralization in E. coli biofilms will benefit the development of strategies against diseases involving calcified biofilms as well as the engineering of biofilm-based living composites

Crystal–Chemical and Biological Controls of Elemental Incorporation into Magnetite Nanocrystals

Magnetite nanoparticles possess numerous fundamental, biomedical, and industrial applications, many of which depend on tuning the magnetic properties. This is often achieved by the incorporation of trace and minor elements into the magnetite lattice. Such incorporation was shown to depend strongly on the magnetite formation pathway (i.e., abiotic vs biological), but the mechanisms controlling element partitioning between magnetite and its surrounding precipitation solution remain to be elucidated. Here, we used a combination of theoretical modeling (lattice and crystal field theories) and experimental evidence (high-resolution inductively coupled plasma–mass spectrometry and X-ray absorption spectroscopy) to demonstrate that element incorporation into abiotic magnetite nanoparticles is controlled principally by cation size and valence. Elements from the first series of transition metals (Cr to Zn) constituted exceptions to this finding, as their incorporation appeared to be also controlled by the energy levels of their unfilled 3d orbitals, in line with crystal field mechanisms. We finally show that element incorporation into biological magnetite nanoparticles produced by magnetotactic bacteria (MTB) cannot be explained by crystal–chemical parameters alone, which points to the biological control exerted by the bacteria over the element transfer between the MTB growth medium and the intracellular environment. This screening effect generates biological magnetite with a purer chemical composition in comparison to the abiotic materials formed in a solution of similar composition. Our work establishes a theoretical framework for understanding the crystal–chemical and biological controls of trace and minor cation incorporation into magnetite, thereby providing predictive methods to tailor the composition of magnetite nanoparticles for improved control over magnetic properties

Nonclassical Crystallization Pathway of Transition Metal Phosphate Compounds

Here, we elucidate nonclassical multistep crystallization pathways of transition metal phosphates from aqueous solutions. We followed precipitation processes of M-struvites, NH4MPO4·6H2O, and M-phosphate octahydrates, M3(PO4)2·8H2O, where M = Ni, Co, or NixCo1–x, by using in situ scattering and spectroscopy-based techniques, supported by elemental mass spectrometry analyses and advanced electron microscopy. Ni and Co phosphates crystallize via intermediate colloidal amorphous nanophases, which change their complex structures while agglomerating, condensing, and densifying throughout the extended reaction times. We reconstructed the three-dimensional morphology of these precursors by employing cryo-electron tomography (cryo-ET). We found that the complex interplay between metastable amorphous colloids and protocrystalline units determines the reaction pathways. Ultimately, the same crystalline structure, such as struvite, is formed. However, the multistep process stages vary in complexity and can last from a few minutes to several hours depending on the selected transition metal(s), their concentration, and the Ni/Co ratio

Nonclassical Crystallization Pathway of Transition Metal Phosphate Compounds

Here, we elucidate nonclassical multistep crystallization pathways of transition metal phosphates from aqueous solutions. We followed precipitation processes of M-struvites, NH4MPO4·6H2O, and M-phosphate octahydrates, M3(PO4)2·8H2O, where M = Ni, Co, or NixCo1–x, by using in situ scattering and spectroscopy-based techniques, supported by elemental mass spectrometry analyses and advanced electron microscopy. Ni and Co phosphates crystallize via intermediate colloidal amorphous nanophases, which change their complex structures while agglomerating, condensing, and densifying throughout the extended reaction times. We reconstructed the three-dimensional morphology of these precursors by employing cryo-electron tomography (cryo-ET). We found that the complex interplay between metastable amorphous colloids and protocrystalline units determines the reaction pathways. Ultimately, the same crystalline structure, such as struvite, is formed. However, the multistep process stages vary in complexity and can last from a few minutes to several hours depending on the selected transition metal(s), their concentration, and the Ni/Co ratio

Nonclassical Crystallization Pathway of Transition Metal Phosphate Compounds

Here, we elucidate nonclassical multistep crystallization pathways of transition metal phosphates from aqueous solutions. We followed precipitation processes of M-struvites, NH4MPO4·6H2O, and M-phosphate octahydrates, M3(PO4)2·8H2O, where M = Ni, Co, or NixCo1–x, by using in situ scattering and spectroscopy-based techniques, supported by elemental mass spectrometry analyses and advanced electron microscopy. Ni and Co phosphates crystallize via intermediate colloidal amorphous nanophases, which change their complex structures while agglomerating, condensing, and densifying throughout the extended reaction times. We reconstructed the three-dimensional morphology of these precursors by employing cryo-electron tomography (cryo-ET). We found that the complex interplay between metastable amorphous colloids and protocrystalline units determines the reaction pathways. Ultimately, the same crystalline structure, such as struvite, is formed. However, the multistep process stages vary in complexity and can last from a few minutes to several hours depending on the selected transition metal(s), their concentration, and the Ni/Co ratio

Nonclassical Crystallization Pathway of Transition Metal Phosphate Compounds

Here, we elucidate nonclassical multistep crystallization pathways of transition metal phosphates from aqueous solutions. We followed precipitation processes of M-struvites, NH4MPO4·6H2O, and M-phosphate octahydrates, M3(PO4)2·8H2O, where M = Ni, Co, or NixCo1–x, by using in situ scattering and spectroscopy-based techniques, supported by elemental mass spectrometry analyses and advanced electron microscopy. Ni and Co phosphates crystallize via intermediate colloidal amorphous nanophases, which change their complex structures while agglomerating, condensing, and densifying throughout the extended reaction times. We reconstructed the three-dimensional morphology of these precursors by employing cryo-electron tomography (cryo-ET). We found that the complex interplay between metastable amorphous colloids and protocrystalline units determines the reaction pathways. Ultimately, the same crystalline structure, such as struvite, is formed. However, the multistep process stages vary in complexity and can last from a few minutes to several hours depending on the selected transition metal(s), their concentration, and the Ni/Co ratio