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 exo-polysaccharide (EPS) and the fibre protein TasA. In monocultures, Delta eps and Delta tasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale and Delta tasA gradually outcompetes its partner Delta eps. Pellicle formation can evolve independently from division of labour in Delta eps and Delta 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 (Delta eps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochemical properties of the protein. Conversely, the EPS producer (Delta tasA) 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 Delta eps and Delta tasA 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.