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.

Organisms as simple as bacteria can engage in complex collective actions, such as group motility and 28 fruiting body formation. Some of these actions involve a division of labor, where phenotypically 29 specialized clonal subpopulations, or genetically distinct lineages cooperate with each other by 30 performing complementary tasks. Here, we combine experimental and computational approaches to 31 investigate any benefits arising from division of labor during biofilm matrix production. We show that 32 both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation 33 in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components; 34 EPS and TasA. We observed that clonal groups of B. subtilis phenotypically segregate in three 35 subpopulations composed of matrix non-producers, EPS-producers, and generalists, which produce 36 both EPS and TasA. We further found that this incomplete phenotypic specialization was 37 outperformed by a genetic division of labor, where two mutants, engineered as strict specialists, 38 . CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/237230 doi: bioRxiv preprint first posted online Dec. 21, 2017; 2 complemented each other by exchanging EPS and TasA. The relative fitness of the two mutants 39 displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency 40 reaching an evolutionary stable equilibrium at 30% TasA-producers, corresponding exactly to the 41 population composition where group fitness is maximized. Using individual-based modelling, we could 42show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix 43 compounds generate for the collective; and that genetic division of labor can be favored when it 44 breaks metabolic constraints associated with the simultaneous production of two matrix components. Microbes can act collectively in groups, and thereby substantially influence their local environment in 62 their own benefit. Such beneficial collective actions include the secretion of nutrient-degrading 63 enzymes [1,2], iron-scavenging siderophores [3], biosurfactants for group motility [4,5], and structural 64 components for biofilm formation [6,7]. In certain cases, cooperation even involves a division of labor, 65where subpopulations of cells specialize to perform different tasks [8][9][10]. For instance, during sliding 66 colony expansion Bacillus subtilis cells phenotypically differentiate into surfactant producers and 67 matrix producers where the role of the first is to reduce surface tension, while the latter allows 68 expanding colony 'arms' to form and explore new territories [10]. Given the high relatedness between 69 cells, specialization is likely beneficial for the group as a whole [11,12], with individuals gaining an 70 inclusive fitness benefit from helping their clone ...

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.