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.

No abstract

In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve the collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells both in biofilms and in multicellular eukaryotes are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review have provided a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development.

Bacillus subtilis biofilms have a fundamental role in shaping the soil ecosystem. During this process, they unavoidably interact with neighbour bacterial species. We studied the interspecies interactions between biofilms of the soil-residing bacteria B. subtilis and related Bacillus species. We found that proximity between the biofilms triggered recruitment of motile B. subtilis cells, which engulfed the competing Bacillus simplex colony. Upon interaction, B. subtilis secreted surfactin and cannibalism toxins, at concentrations that were inert to B. subtilis itself, which eliminated the B. simplex colony, as well as colonies of Bacillus toyonensis. Surfactin toxicity was correlated with the presence of short carbon-tail length isomers, and synergistic with the cannibalism toxins. Importantly, during biofilm development and interspecies interactions a subpopulation in B. subtilis biofilm lost its native plasmid, leading to increased virulence against the competing Bacillus species. Overall, these findings indicate that genetic programs and traits that have little effect on biofilm development when each species is grown in isolation have a dramatic impact when different bacterial species interact.

In nature, bacteria form biofilms—differentiated multicellular communities attached to surfaces. Within these generally sessile biofilms, a subset of cells continues to express motility genes. We found that this subpopulation enabled Bacillus subtilis biofilms to expand on high-friction surfaces. The extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA acted as a developmental signal stimulating a subset of biofilm cells to revert to a motile phenotype. Transcriptomic analysis revealed that TasA stimulated the expression of a specific subset of genes whose products promote motility and repress ECM production. Spontaneous suppressor mutations that restored motility in the absence of TasA revealed that activation of the biofilm-motility switch by the two-component system CssR/CssS antagonized the TasA-mediated reversion to motility in biofilm cells. Our results suggest that although mostly sessile, biofilms retain a degree of motility by actively maintaining a motile subpopulation.