The formation of microbial exoskeletons is driven by a controlled calcium-concentrating subcellular niche

Abstract

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 ...

Calcium promotes persistent soil organic matter by altering microbial transformation of plant litter

“…The abiotic and biotic factors driving changes in bacterial community composition, and the prevalence of surface-colonizing populations, cannot be disentangled using phylogenetic gene marker data, and are likely a result of a combination of factors (see Supplementary Information for more details). One likely contributing factor is the direct effect of Ca, which is often a critical co-factor in bacterial attachment and biofilm production [20][21][22][23] , promoting the growth and activity of surface-adhering bacterial populations. The reduction in mineralization in Ca-treated soils likely reflect physiological and metabolic differences in surface-attached populations compared to bacteria adapted for growth in pore water, including slower growth rates, as well as increased association between microbial byproducts (e.g., adhesive proteins and extracellular polymeric substances) and mineral surfaces 48 .…”

“…The current paradigm for how Ca affects SOC persistence overlooks its potential influence over the first step, the microbial transformation of plant C.The fate of plant and soil C is affected by its composition, C:N ratio, the microbial C use efficiency (CUE), and abiotic conditions (e.g., soil water content, mineralogy, and pH) [15][16][17] . The microbial processing of plant C can also be impacted by soil Ca contents 18,19 , since Ca is a key factor in the growth and activity of fungi and bacteria, in particular surface-adhering [20][21][22] and biofilm-forming bacteria, [20][21][22][23] as well as fungal lignin-degrading enzymes 24 . Enrichment of bacterial taxa that attach to particulate C sources (i.e., plant litter or MAOM) may limit SOC gain due to enhanced litter mineralization 19 , or conversely, may enhance SOC gain by limiting litter decomposition 18 .…”

Calcium promotes persistent soil organic matter by altering microbial transformation of plant litter

“…The selection for surface-adhering bacterial populations can be explained by the fact that Ca is a critical co-factor in many modes of bacterial attachment (see Supplementary Information for more details) and bio lm production [20][21][22][23] . We anticipate that surface-attached populations will exhibit differences in metabolism from those bacteria better adapted for growth in pore water, favoring slower growth rates, higher CUE, and an increase in microbial products that facilitate surface interactions (e.g., adhesive proteins and extracellular polymeric substances).…”

“…The current paradigm for how Ca affects SOC accumulation overlooks its potential in uence over the rst step, microbial transformation of plant C, which is affected by its C:N, the microbial C use e ciency (CUE), and abiotic conditions (e.g., soil moisture and pH) [15][16][17] . Plant C transformation may also be impacted by soil Ca content 18,19 , since Ca is a key factor in microbial surface adhesion, such as to particulate organic substrates and soil minerals, bio lm development 20 , and growth of surface-associated bacteria [21][22][23] . Enrichment in bacterial taxa that attach to particulate C sources (i.e., plant litter or MAOM) may limit SOC gain due to enhanced litter mineralization 19 , or conversely, may enhance SOC gain by limiting litter decomposition 18 .…”

Extracellular Ca2+-sensitive fluorescent protein biosensor based on a collagen-binding domain

“…In addition to the physical tension [4] and the extracellular matrix itself [5], changing cell density, composition and organization can also affect gradients of O2 (hypoxia) [6,7], distribution and diffusion of reactive oxygen species, peptides and growth factors, pH and other ions such as Ca 2+ [8,9]. A number of reports have indicated the role of extracellular Ca 2+ as one the primary signals that influence cell function [10]: thus, regulation of extracellular Ca 2+ have been shown to be relevant to gastrointestinal tract function [11,12], bone marrow [13], brain and CNS function [14, 15], smooth and skeletal muscles [16], wound healing [17], plasma membrane repair [18], engineered organoid-like tissues [19] and microbial biofilm formation [20]. Every mammalian cell relies on the Ca 2+ homeostasis, which in turn is tightly regulated through the activities of channels, ATPases, Na + / Ca 2+ exchanger, cytosolic Ca 2+ -binding proteins and various depots such as endoplasmic reticulum, secretory vesicles and mitochondria [21][22][23][24].…”