37Bacterial biofilms produce a robust internal mineral layer, composed of calcite, 38 which strengthens the colony and protects the residing bacteria from antibiotics. In 39 this work, we provide evidence that the assembly of a functional mineralized 40 macro-structure begins with mineral precipitation within a defined cellular 41 compartment in a differentiated subpopulation of cells. Transcriptomic analysis of 42 a model organism, Bacillus subtilis, revealed that calcium was essential for 43 activation of the biofilm state, and highlighted the role of cellular metal homeostasis 44 and carbon metabolism in biomineralization. The molecular mechanisms 45 promoting calcite formation were conserved in pathogenic Pseudomonas 46 aeruginosa biofilms, resulting in formation of calcite crystals tightly associated with 47 bacterial cells in sputum samples collected from cystic fibrosis patients.
48Biomineralization inhibitors targeting calcium uptake and carbonate accumulation 49 significantly reduced the damage inflicted by P. aeruginosa biofilms to lung tissues.
50Therefore, better understanding of the conserved molecular mechanisms 51 promoting biofilm calcification can path the way to the development of novel 52 classes of antibiotics to combat otherwise untreatable biofilm infections.
54Main text 55 In nature bacteria form differentiated multicellular communities, known as 56 biofilms. Bacterial biofilms are of extreme clinical importance, as they are 57 associated with many persistent and chronic bacterial infections (1). For example, 58 the commensal/aquatic bacterium P. aeruginosa can cause devastating chronic 59 biofilm infections in immune compromised hosts, in patients with cystic fibrosis 60 (CF), and on the surface of medical devices and burn wounds (1). Bacteria in a 61 biofilm can be up to 1,000 times more resistant to antibiotics than planktonic (free-62 living) bacteria (2). The mechanisms supporting this phenotypic resistance, as well 63 as those driving the transition from free-living single bacteria to a differentiated 64 biofilm community are still poorly understood (1, 3). 65 To date, the ability of biofilm-forming bacteria to form complex architectures was 66 attributed exclusively to their organic extracellular matrix (ECM). However, we and 67 others have recently shown that microbial biofilms contain a robust internal 68 mineral layer, composed of crystalline calcium carbonate (calcite) (4-7).
69Calcification associated with biofilms was also observed in clinical settings, such 70 as catheters (8). While the role of bacterial cells as nucleation sites in 71 environmental carbonate mineral formation is long-established (9), it is so far 72 considered an unintentional by-product of bacterial metabolic activity, random 73 and unregulated, and serving no particular function for the mineral-associated 74 bacteria.
75In this work, we provide evidence that the assembly of a functional mineralized 76 macro-structure begins with mineral precipitation within a defined cellular 77 compartment ...