1 Spores and extracellular matrix components impart molecular order in Bacillus subtilis biofilms. David N Azulay 1# , Oliver Späker 2# , Mnar Ghrayeb 1 , Michaela Wilsch-Bräuninger 3 , Ernesto Scoppola 2 , Manfred Burghammer 4 , Ivo Zizak 5 , Luca Bertinetti 6 , Yael Politi 6 * and Liraz Chai 1,8 * 1 Institute of Chemistry, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 91904, Israel 2 Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany 3 Max Planck Institute of Molecular Cell Biology and Genetics, 1307 Dresden, Germany 4 European Synchrotron Radiation Facility (ESRF)71, avenue des Martyrs, CS 40220, Grenoble Cedex 9 38043, France 5 Helmholtz-Zentrum Berlin, Department Structure and Dynamics of Energy Materials, Berlin, Germany 6 B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany 7 The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 91904, Israel # equal contribution * To whom correspondence should be addressed: Dr. Liraz Chai, Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem 91904, Israel. Telephone: +972-2-6585303, Fax. +972-2-5660425. Email. Liraz.chai@mail.huji.ac.il; Dr. Yael Politi, B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, 01307 Dresden, Germany. Email. yael.politi@tu-dresden.de Abstract Biofilms are surface-associated soft microbial communities, which may be either detrimental or beneficial to their hosting environment. They develop from single cells into mature colonies, that are composed of cells and sometimes (in Firmicutes phylum) spores, held together by an extracellular matrix (ECM) of secreted biomolecular components. Biofilm development is a dynamic process, during which cells organize into subgroups, creating functionally distinct regions in space. Specific examples of functional-spatial division in Bacillus subtilis biofilms include matrix and spore formation as well as water channels that form beneath wrinkles. An interesting question arising is whether the division of labor in biofilms is also reflected in the molecular-level order across whole biofilms. Using combined X-ray diffraction (XRD)/X-ray fluorescence (XRF), we studied the molecular order in intact biofilms across multiple length scales. We discovered that biofilms display a distinct spatio-temporal XRD signature that depends on highly ordered structures in spores and on cross  sheet structures in matrix components. Spore signal is found especially enhanced with water molecules and metal-ions signals along macroscopic wrinkles, known to act as water channels. Demonstrating in situ the link between molecular structures, metal ions distribution and division of labor across whole biofilms in time and space, this study provides new pivotal insight to the understanding biofilm development. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted July 27, 2021. ; https://doi.org/10.1101/2021.07.27.453653 doi: bioRxiv preprint