Contents lists available at ScienceDirect New BIOTECHNOLOGY journal homepage: www.elsevier.com/locate/nbt Review Article A brick in the wall: Discovering a novel mineral component of the biofilm extracellular matrix Alona Keren-Paz, Ilana Kolodkin-Gal* Department of Molecular Genetics, Weizmann Institute of Science, 234 Herzl Street, Rehovot, 76100, Israel ARTICLE INFO Keywords: Biofilms Extracellular Matrix Biomineralization Persistent Infections ABSTRACT Multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self- produced organic extracellular matrix (ECM). However, new evidence for a missed mineral constituent of ECM in both Gram-positive and Gram-negative bacterial species, is accumulating. Study of two phylogenetically distinct bacteria, Bacillus subtilis and Mycobacterium smegmatis, identified a novel mechanism crucial for proper biofilm development and architecture – an active, genetically regulated, production of crystalline calcite. The calcite scaffolds stabilize bacterial biofilms, limit penetration of small molecule solutes such as antibiotics and play a conserved role in the assembly of those complex differentiated multicellular communities. This review discusses the recently discovered structural and functional roles of extracellular minerals in biofilms. It is pro- posed that it is time for a more complete view of the ECM as a complex combination of organic and nonorganic materials, especially in the light of the possible implications for treatment of biofilm infections. Introduction While historically thought of as unicellular organisms, in nature bacteria form complex and differentiated multicellular communities, known as biofilms. The coordinated actions of many cells, commu- nicating and dividing labour, improve the ability of the community to attach to hosts and protect it from environmental assaults [1,2]. Bac- terial biofilms are of extreme clinical importance, as they are inherently associated with many persistent and chronic bacterial infections [3]. For example, the commensal/aquatic bacterium Pseudomonas aerugi- nosa can cause devastating chronic biofilm infections in compromised hosts, including cystic fibrosis (CF) patients, on medical devices and burn wounds [3]. Moreover, biofilms are inherently resistant to anti- biotics [4,5]. In a biofilm, bacteria can be up to 1000 times more re- sistant to antibiotics than when planktonic (free-living) [4]. The me- chanisms supporting this phenotypic resistance, as well as those driving the transition from free-living single bacteria to differentiated biofilm community, are still poorly understood. It is now clear that effective control of biofilm-related infections will require a concerted effort to develop therapeutic agents that prevent the formation, or promote the detachment, of the biofilms [3]. The single cells composing a biofilm often exhibit a well-defined spatial organization [6–8]. The 3D structure of the biofilm has been suggested to relieve metabolic stress. For example, channels formed below the ridges and wrinkles within the colony may facilitate diffusion of fluids, nutrients and oxygen [9–12]. In addition, cells residing in different areas of the colony are exposed to different levels of O 2 , nu- trients and quorum sensing molecules, thereby affecting the genetic programs they express [6,13–18]. To date, the ability of biofilm-forming bacteria to form complex architectures has been exclusively attributed to their organic extra- cellular matrix (ECM). However, it was shown recently that biofilm colonies can also contain a robust internal mineral layer, composed of CaCO 3 (Fig. 1)[19]. It is spatially organized, forms during biofilm development in at least two distinct model organisms (Bacillus subtilis and Mycobacterium smegmatis, both related to several important pa- thogens), and thus might be a general phenomenon common to many distinct bacterial species [19]. Bacterial genes and environmental triggers have been identifed that contribute to this biomineralization process. The results indicate that this mineral component strengthens biofilm architecture and serves as a framework to support larger bac- terial populations. In addition, this mineral structure protects bacteria from antibiotics. This report was not the first to demonstrate calcifi- cation in clinical biofilms, which were first observed in the late 1990s on catheters [20]. However, the first demonstrations that these mineral scaffolds are tightly regulated by the biofilm cells, and a possible https://doi.org/10.1016/j.nbt.2019.11.002 Abbreviations: AHA, acetohydroxamic acid; BCM, biologically controlled biomineralization; BIM, biologically induced biomineralization; CF, cystic fibrosis; ECM, extracellular matrix; eDNA, extracellular DNA; FTIR, Fourier-transform infrared spectroscopy ⁎ Corresponding author. E-mail address: ilana.kolodkin-gal@weizmann.ac.il (I. Kolodkin-Gal). New BIOTECHNOLOGY 56 (2020) 9–15 Available online 06 November 2019 1871-6784/ © 2019 Published by Elsevier B.V.