Historically, multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self-produced extracellular matrix. Our study identified a novel mechanism maintaining Bacillus subtilis and Mycobacterium smegmatis biofilms—active production of calcite minerals. We studied, for the first time, the effects of mutants defective in biomineralization and calcite formation on biofilm development, resilience and morphology. We demonstrated that an intrinsic rise in carbon dioxide levels within the biofilm is a strong trigger for the initiation of calcite-dependent patterning. The calcite-dependent patterns provide resistance to environmental insults and increase the overall fitness of the microbial community. Our results suggest that it is highly feasible that the formation of mineral scaffolds plays a cardinal and conserved role in bacterial multicellularity.
Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered a heterogeneous response in LAB strains, frequently manifested specifically in altered carrying capacity during planktonic growth and colony development. The fermentation capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamanosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation. We suggest that the optimal performance of the beneficial microbiota members belonging to lactobacilli is heterogeneous and varies as a function of the growth model and the dependency on a buffering system.
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 ...
Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth. In addition, heterogeneous response to fermentable sugar was manifested in microbial aggregation (measured by imaging flow cytometry), colony development, and attachment to mucin. The acid production capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamnosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation, but the formation of biofilms and aggregation capacity were responsive to the carbohydrate provided rather than to the acidity. We suggest that the optimal performance of the beneficial microbiota members belonging to Lactobacillaceae varies as a function of the growth model and the dependency on a buffering system.
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Lacticaseibacillus rhamnosus GG(LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates (e.g., glucose, mannose) for energy production under conditions of oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture in LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced secretion of known and novel metabolites and proteins targeting the enteric pathogensEnterococcus faecalisandSalmonella Enterica serovar Typhimurium. Overall, our results link the common metabolic regulation of cell wall remodeling, aggregation to host tissues, biofilm formation in probiotic strains, and connect the production of antimicrobial effectors with metabolome reprogramming. These findings provide novel insights into the role of nutrition in the establishment of LGG in the gastrointestinal tract.
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