Biofilms are biological living tissues that appear when bacteria colonize a surface and synthesize extracellular matrix components, as a survival strategy in challenging environments. In addition to the organic matrix, some biofilms are able to accumulate mineral particles such as calcium phosphate, which is also widespread in eukaryotes, e.g. as mineral phase of their skeleton. In bone, alkaline phosphatase (ALP) plays a key role in calcium phosphate precipitation. Indeed, this enzyme catalyzes the hydrolysis of monophosphates starting from different precursors (e.g., alkaloids, proteins) and makes phosphate ions readily available for the interaction with cations (e.g., calcium). ALP is not only present in eukaryotic cells, but also in the periplasmic membrane of many prokaryotes such as E. coli, S. aureus and various soil bacteria, where it is responsible for several cellular processes (e.g. protein phosphorylation, cell growth and differentiation) [1]. While it has been recently speculated that bacteria can actively induce mineral precipitation exploiting this enzyme [2], the role of ALP in matrix production and mineralization is not fully understood [1]. In this context, we aim to shed some light on the mechanism of E. coli biofilm mineralization. As a model, we chose the biofilm-forming strain E. coli K-12 W3110, which possesses periplasmic alkaline phosphatase [3]. We investigated the mineralization conditions of biofilms grown on nutritive agar substrates and localized the mineral phase through light and scanning electron microscopy. X-ray powder diffractometry and wide-angle x-ray scattering enabled to identify the mineral as being hydroxyapatite. Finally, growing the E. coli bacteria on mineralizing medium supplemented with an ALP-inhibitor confirmed that the presence of active ALP is essential to biofilm mineralization. Uncovering the mineralization mechanisms in biofilms, is an important piece of knowledge to defy diseases related to calcified biofilms like periodontitis, but it can be also exploited to engineer living composites, such as self-repairing materials.


References
[1] K. M. Danikowski, T. Cheng (2019) J Vis Exp 2019, 146, 2–5.
[2] S. Omelon, M. Ariganello, E. Bonucci, et al. Calcif Tissue Int, 2013, 93, 382-396
[3] K.L. Rogers, J. Cosmidis, K. Benzerara, et al. Front Earth Sci, 2015, 3, 1–20.