The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging

Abstract

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

“…Furthermore, the attachment on PS and SS were comparable, which indicates that a denser biofilm formation on SS cannot be explained by a higher initial attachment. Similar findings were reported previously showing no correlation between the initial attachment by different strains of B. subtilis to PS within 1.5 h and the biofilm formed after 48 h (Bridier et al 2011).…”

Bacterial swimmers that infiltrate and take over the biofilm matrix

“…Swimming activity was also tested in a biofilm comprising a B. subtilis 168 ΔabrB mutant, in which matrix components and biofilm formation are up-regulated (14,15). The large clusters formed by this mutant were refractive to B. thuringiensis swimmers.…”

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

“…However, submerged biofilms are the most widely used model to study the formation of biofilms and their properties for pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus (25,26). While the submerged model represents environmental conditions confronted by B. subtilis in its natural habitats, it has only recently been applied to the species (22,27), likely due to experimental limitations associated with the bacterium (17,27). In a previous study, we proposed a new methodology to visualize and quantify B. subtilis submerged biofilms using a microplate-based model combined with confocal laser scanning microscopy (CLSM) (27,28).…”

“…While the submerged model represents environmental conditions confronted by B. subtilis in its natural habitats, it has only recently been applied to the species (22,27), likely due to experimental limitations associated with the bacterium (17,27). In a previous study, we proposed a new methodology to visualize and quantify B. subtilis submerged biofilms using a microplate-based model combined with confocal laser scanning microscopy (CLSM) (27,28). This experimental system enabled the identification of a remarkable B. subtilis strain (NDmed), isolated from an endoscope washer-disinfector (29), capable of forming thick and protruding biofilms highly resistant to the biocides commonly used for endoscope disinfection in hospitals (30).…”