Induced mineralization in Escherichia coli biofilms: the key 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. The enzyme alkaline phosphatase (ALP) plays a key role in calcium phosphate precipitation in mammalian bone tissue. Produced by eukaryotic cells, ALP catalyzes the hydrolysis of monophosphates starting from different precursors (e.g., alkaloids, proteins) and makes phosphate ions readily available for the precipitation with calcium. Bacterial ALPs are expressed by the well-characterized gram-negative and gram-positive bacteria E. coli and S. aureus as well as a large number of marine and soil bacteria. While it was recently proposed that bacterial ALPs induce mineral precipitation, their role in biofilm mineralization is not fully understood. In this work, we address this question using the biofilm-forming E. coli K-12 strain W3110, which expresses periplasmic ALP from the phoA gene. We first identify the mineralization conditions of biofilms grown on nutritive agar substrates supplemented with calcium ions and {beta}-glycerophosphate. We then localize the mineral phase at different scales, using light and scanning electron microscopy as well as X-ray microtomography. Wide-angle X-ray scattering enables us to further identify the mineral as being hydroxyapatite. Finally, growing E. coli cells on mineralizing medium supplemented with an ALP inhibitor demonstrates that ALP is essential for biofilm mineralization. This is confirmed with a bacteria-free model, where the deposition of a drop of bacterial ALP solution on calcium and {beta}-glycerophosphate containing agar substrate is sufficient to induce mineralization. Overall, these results will benefit the development of strategies against diseases involving calcified biofilms as well as the engineering of biofilm-based living composites.