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.