Privatization of Biofilm Matrix in Structurally Heterogeneous Biofilms.

The self-produced biofilm provides beneficial protection for the enclosed cells, but the costly production of matrix components makes producer cells susceptible to cheating by nonproducing individuals. Despite detrimental effects of nonproducers, biofilms can be heterogeneous, with isogenic nonproducers being a natural consequence of phenotypic differentiation processes. For instance, in Bacillus subtilis biofilm cells differ in production of the two major matrix components, the amyloid fiber protein TasA and exopolysaccharides (EPS), demonstrating different expression levels of corresponding matrix genes. This raises questions regarding matrix gene expression dynamics during biofilm development and the impact of phenotypic nonproducers on biofilm robustness. Here, we show that biofilms are structurally heterogeneous and can be separated into strongly and weakly associated clusters. We reveal that spatiotemporal changes in structural heterogeneity correlate with matrix gene expression, with TasA playing a key role in biofilm integrity and timing of development. We show that the matrix remains partially privatized by the producer subpopulation, where cells tightly stick together even when exposed to shear stress. Our results support previous findings on the existence of "weak points" in seemingly robust biofilms as well as on the key role of linkage proteins in biofilm formation. Furthermore, we provide a starting point for investigating the privatization of common goods within isogenic populations.IMPORTANCE Biofilms are communities of bacteria protected by a self-produced extracellular matrix. The detrimental effects of nonproducing individuals on biofilm development raise questions about the dynamics between community members, especially when isogenic nonproducers exist within wild-type populations. We asked ourselves whether phenotypic nonproducers impact biofilm robustness, and where and when this heterogeneity of matrix gene expression occurs. Based on our results, we propose that the matrix remains partly privatized by the producing subpopulation, since producing cells stick together when exposed to shear stress. The important role of linkage proteins in robustness and development of the structurally heterogeneous biofilm provides an entry into studying the privatization of common goods within isogenic populations.

Cheaters shape the evolution of phenotypic heterogeneity in Bacillus subtilis biofilms.

Biofilms are closely packed cells held and shielded by extracellular matrix composed of structural proteins and exopolysaccharides (EPS). As matrix components are costly to produce and shared within the population, EPS-deficient cells can act as cheaters by gaining benefits from the cooperative nature of EPS producers. Remarkably, genetically programmed EPS producers can also exhibit phenotypic heterogeneity at single-cell level. Previous studies have shown that spatial structure of biofilms limits the spread of cheaters, but the long-term influence of cheating on biofilm evolution is not well understood. Here, we examine the influence of EPS nonproducers on evolution of matrix production within the populations of EPS producers in a model biofilm-forming bacterium, Bacillus subtilis. We discovered that general adaptation to biofilm lifestyle leads to an increase in phenotypical heterogeneity of eps expression. However, prolonged exposure to EPS-deficient cheaters may result in different adaptive strategy, where eps expression increases uniformly within the population. We propose a molecular mechanism behind such adaptive strategy and demonstrate how it can benefit the EPS producers in the presence of cheaters. This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.

Publisher Correction: Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms.

In the version of this Article originally published, author Carolina Falcón Garcia's name was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.

Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms.

Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain 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. Conversely, 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 versus exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an additional barrier to evolution of division of labour between genetically distinct microorganisms.

Division of Labor during Biofilm Matrix Production.

Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components, exopolysaccharides (EPSs) and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS producers, and generalists, which produce both EPSs and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPSs and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modeling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components.

Evolution of exploitative interactions during diversification in Bacillus subtilis biofilms.

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 discovered 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.

De novo evolved interference competition promotes the spread of biofilm defectors.

Biofilms are social entities where bacteria live in tightly packed agglomerations, surrounded by self-secreted exopolymers. Since production of exopolymers is costly and potentially exploitable by non-producers, mechanisms that prevent invasion of non-producing mutants are hypothesized. Here we study long-term dynamics and evolution in Bacillus subtilis biofilm populations consisting of wild-type (WT) matrix producers and mutant non-producers. We show that non-producers initially fail to incorporate into biofilms formed by the WT cells, resulting in 100-fold lower final frequency compared to the WT. However, this is modulated in a long-term scenario, as non-producers evolve the ability to better incorporate into biofilms, thereby slightly decreasing the productivity of the whole population. Detailed molecular analysis reveals that the unexpected shift in the initially stable biofilm is coupled with newly evolved phage-mediated interference competition. Our work therefore demonstrates how collective behaviour can be disrupted as a result of rapid adaptation through mobile genetic elements.

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins. Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

Laboratory Evolution of Microbial Interactions in Bacterial Biofilms.

Microbial adaptation is conspicuous in essentially every environment, but the mechanisms of adaptive evolution are poorly understood. Studying evolution in the laboratory under controlled conditions can be a tractable approach, particularly when new, discernible phenotypes evolve rapidly. This is especially the case in the spatially structured environments of biofilms, which promote the occurrence and stability of new, heritable phenotypes. Further, diversity in biofilms can give rise to nascent social interactions among coexisting mutants and enable the study of the emerging field of sociomicrobiology. Here, we review findings from laboratory evolution experiments with either Pseudomonas fluorescens or Burkholderia cenocepacia in spatially structured environments that promote biofilm formation. In both systems, ecotypes with overlapping niches evolve and produce competitive or facilitative interactions that lead to novel community attributes, demonstrating the parallelism of adaptive processes captured in the lab.