Here, we review the multiple mechanisms that the Gram-positive bacterium Bacillus subtilis uses to allow it to communicate between cells and establish community structures. The modes of action that are used are highly varied and include routes that sense pheromone levels during quorum sensing and control gene regulation, the intimate coupling of cells via nanotubes to share cytoplasmic contents, and long-range electrical signalling to couple metabolic processes both within and between biofilms. We explore the ability of B. subtilis to detect 'kin' (and 'cheater cells') by looking at the mechanisms used to potentially ensure beneficial sharing (or limit exploitation) of extracellular 'public goods'. Finally, reflecting on the array of methods that a single bacterium has at its disposal to ensure maximal benefit for its progeny, we highlight that a large future challenge will be integrating how these systems interact in mixed-species communities.

Biofilm formation by Bacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm into a nonexpanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that B. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe3+ from the growth medium in a nonenzymatic reaction. Utilizing mathematical modeling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated with pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion by neighboring communities through the generation of an “iron-free” zone, thereby addressing the paradox of pulcherriminic acid production by B. subtilis.

Bacteria can form dense communities called biofilms, where cells are embedded in a self-produced extracellular matrix. Exploiting competitive interactions between strains within the biofilm context can have potential applications in biological, medical, and industrial systems. By combining mathematical modelling with experimental assays, we reveal that spatial structure and competitive dynamics within biofilms are significantly affected by the location and density of the founder cells used to inoculate the biofilm. Using a species-independent theoretical framework describing colony biofilm formation, we show that the observed spatial structure and relative strain biomass in a mature biofilm comprising two isogenic strains can be mapped directly to the geographical distributions of founder cells. Moreover, we define a predictor of competitive outcome that accurately forecasts relative abundance of strains based solely on the founder cells’ potential for radial expansion. Consequently, we reveal that variability of competitive outcome in biofilms inoculated at low founder density is a natural consequence of the random positioning of founding cells in the inoculum. Extension of our study to non-isogenic strains that interact through local antagonisms, shows that even for strains with different competition strengths, a race for space remains the dominant mode of competition in low founder density biofilms. Our results, verified by experimental assays using Bacillus subtilis, highlight the importance of spatial dynamics on competitive interactions within biofilms and hence to related applications.

Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our novel isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason for the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

Bacteria typically form dense communities called biofilms, where cells are embedded in a self-produced extracellular matrix. Competitive interactions between strains within the biofilm context are studied due to their potential applications in biological, medical, and industrial systems. Combining mathematical modelling with experimental assays, we reveal that the spatial structure and the competitive dynamics within biofilms are significantly affected by the location and density of founder cells. Using an isogenic pair of Bacillus subtilis strains, we show that the observed spatial structure and relative strain biomass in a mature biofilm can be mapped directly to the locations of founder cells. Moreover, we define a predictor of competitive outcome that accurately forecasts relative abundance of strains based solely on the founder cells’ access to free space. Consequently, we reveal that variability of competitive outcome in biofilms inoculated at low founder density is a natural consequence of the random positioning of founding cells in the inoculum. Extending our study to non-isogenic strain pairs of B. subtilis, we show that even for strains with different antagonistic strengths, a race for space remains the dominant mode of competition in biofilms inoculated at low founder densities. Our results highlight the importance of spatial dynamics on competitive interactions within biofilms and hence to related applications.