Laura Zorzetto (1), Ernesto Scoppola (1), Emeline Raguin (1), Kerstin G. Blank (2) (3), Peter Fratzl (1), Cécile M. Bidan (1)
Chemistry of Materials. Volume 35, Issue 13 (31 March 2023). DOI: https://doi.org/10.1021/acs.chemmater.2c02969

Keywords

Bacteria, biofilms, calcium, mineralization, minerals

Abstract

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 colibiofilms 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.

How Our Software Was Used

Stack alignment, image processing, segmentation, and video generation were done using Dragonfly. BSE and mixed Inlens/SE images were first automatically aligned using the sum of squared difference (SSD) method available in the Slice Registration panel. Curtaining artifacts produced by the beam during the milling process were corrected using a vertical destriping filter. A gradient domain fusion filter was applied only to the mixed Inlens/SE images in order to attenuate the discontinuities in the illumination from registered image stacks and further image filtering was applied to improve contrast and reduce noise. Segmentation was performed with Dragonfly’s Deep Learning module.

Author Affiliation

(1) Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany
(2) Mechano(bio)chemistry, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany
(3) Institute of Experimental Physics, Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz, Austria