The colonization of surfaces by bacterial biofilms constitutes a huge problem in healthcare and industry. When attempting biofilm inactivation or removal, it is crucial to sufficiently wet the biofilm surface with antibacterial agents; however, certain biofilms efficiently resist wetting, and the origin of this behavior remains to date unclear. Here, we demonstrate that, depending on the growth medium used, the model bacterium Bacillus subtilis can form biofilm colonies with distinct surface properties: we find either hydrophilic or two variants of hydrophobic behavior. We show that those differences in biofilm wetting correlate with distinct surface topologies which, in turn, give rise to different physical wetting regimes known from lotus leaves or rose petals. Forming biofilms with different wetting properties may help bacteria to survive in both arid and humid conditions. Furthermore, converting the surface polarity of a biofilm could facilitate their removal from surfaces by increasing their wettability.

Closely related microbes often cooperate, but the prevalence and stability of cooperation between different genotypes remains debatable. Here, we track the evolution of pellicle biofilms formed through genetic division of labour and ask whether partially deficient partners can evolve autonomy.Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exopolysaccharide (EPS) and the fibre protein TasA. In monocultures, ∆eps and ∆tasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale, and ∆tasA gradually outcompetes its partner ∆eps. Pellicle formation can evolve independently from division of labour in ∆eps and ∆tasA monocultures, by selection acting on the residual matrix component, TasA or EPS respectively. Using a set of interdisciplinary tools, we unravel that the TasA-producer (∆eps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochemical properties of the protein. On the contrary, the EPS-producer (ΔtasA) undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomechanical properties. Finally, we revisit the collapse of division of labour between Δeps and ΔtasA in light of a strong frequency vs. exploitability trade-off that manifested in the solitarily evolving partners. We propose such trade-off differences may represent an additional barrier to evolution of division of labour between genetically distinct microbes.

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Surface superhydrophobicity makes bacterial biofilms very difficult to fight, and it is a combination of their matrix composition and complex surface roughness which synergistically protects these biomaterials from wetting. Although trying to eradicate biofilms with aqueous (antibiotic) solutions is common practice, this can be a futile approach if the biofilms have superhydrophobic properties. To date, there are not many options available to reduce the liquid repellency of biofilms or to prevent this material property from developing. Here, we present a solution to this challenge. We demonstrate how the addition of metal ions such as copper and zinc during or after biofilm formation can render the surface of otherwise superhydrophobic B. subtilis NCIB 3610 biofilms completely wettable. As a result of this procedure, these smoother, hydrophilic biofilms are more susceptible to aqueous antibiotics solutions. Our strategy proposes a scalable and widely applicable step in a multi-faceted approach to eradicate biofilms.

Treatment with concentrated ethanol, saline or glucose solutions smoothens biofilm surface topography and initially superhydrophobic/omniphobic biofilms are rendered hydrophilic.

The production of biomolecules can provide new functionalities to the synthesizing organism. One important example is the secretion of extracellular polymeric substances (EPS) by biofilm forming bacteria. This biofilm matrix protects the individual bacteria within the biofilm from external stressors such as antibiotics, chemicals and shear flow. Previous studies have determined several main matrix components of biofilms formed by Bacillus subtilis. However, how these matrix components influence the growth behavior and final dimensions of B. subtilis biofilms has not yet been determined. Here, we combine different experimental techniques with theoretical modeling to assess this relation. In particular, we quantify the area covered by the biofilm and the biofilm height by performing time-lapse microscopy and light profilometry, respectively. We study the development of biofilms formed by two wild-type strains (B-1 and NCIB 3610) differing in their matrix composition and NCIB 3610 mutant strains lacking the ability to produce specific EPS. Based on the experimentally obtained growth dynamics, we develop a mathematical model that allows us to quantify the influence of three key biofilm matrix components on the final NCIB 3610 biofilm colony dimensions. In detail, we show that two matrix components, the exopolysaccharide produced by the epsA-O operon and the surface layer protein BslA control the area covered by the biofilm colony. The height of these mature biofilm colonies is mostly affected by BslA.Together, our results emphasize the importance of the biofilm matrix composition for biofilm growth and the final dimensions of mature B. subtilis NCIB 3610 biofilm colonies.

Microbial biofilms are tightly packed, heterogeneous structures that serve as arenas for social interactions. Studies on Gram negative models reveal that during evolution in structured environments like biofilms, isogenic populations commonly diversify into phenotypically and genetically distinct variants. These variants can settle in alternative biofilm niches and develop new types of interactions that greatly influence population productivity. Here, we explore the evolutionary diversification of pellicle biofilms of the Gram positive, spore-forming bacterium Bacillus subtilis. We discover that -similarly to other species -B. subtilis diversifies into distinct colony variants. These variants dramatically differ in biofilm formation abilities and expression of biofilm-related genes. In addition, using a quantitative approach, we reveal striking differences in surface complexity and hydrophobicity of the evolved colony types.Interestingly, one of the morphotypes completely lost the ability of independent biofilm formation and evolved to hitchhike with other morphotypes with improved biofilm forming abilities. Genome comparison suggests that major phenotypic transformations between the morphotypes can be triggered by subtle genetic differences. Our work demonstrates how positive complementarity effects and exploitative interactions intertwine during evolutionary diversification in biofilms.

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