Using Bacillus subtilis pellicle (a floating biofilm at an air-liquid interface) formation as a model system, the authors observed that the two major ECM components — EPSs and TasA — are metabolically costly to produce and are shared amongst the community. Using Δeps and ΔtasA mutants in growth competition assays, they observed a significant fitness cost of EPSs and TasA production. In agreement with previous observations, they found that Δeps and ΔtasA mutants could not establish biofilms in monoculture, but could when they were co-cultured or if conditioned media from producers was added, indicating that EPSs and TasA can be shared.
The authors hypothesised that phenotypic differentiation into EPS producers and TasA producers could occur within B. subtilis pellicles. Using confocal laser scanning microscopy and cells expressing fluorescent markers under the control of the ECM gene promoters, they observed heterogeneity in the expression of eps and tasA within pellicles. Specifically, bright clusters of EPS signal were observed in regions where there was reduced TasA signal, suggesting the presence of subpopulations that are specialised for EPS production. Flow cytometry of the pellicle population revealed the presence of three distinct populations of cells: ECM-OFF (EPS and TasA non-producers), ECM-ON (cells that produce both EPS and TasA) and EPS-ON (cells in which an EPS signal was detected only), confirming that wild-type B. subtilis can assume ECM-producing and ECM-non-producing phenotypes. However, this incomplete specialisation (that is, the absence of cells that exclusively produce TasA) does not adhere to the division of labour principle, prompting the authors to investigate whether it is beneficial or whether it can be outperformed by a genetically determined specialisation. By mixing Δeps and ΔtasA mutants at different ratios, pellicle productivity was found to vary in response to strain frequency and a ~30% Δeps frequency resulted in significantly higher pellicle growth in vitro and on the roots of Arabidopsis thaliana than wild-type, demonstrating that genetic division of labour between EPS and TasA producers provides a fitness benefit to biofilm communities.
This is a preview of subscription content, access via your institution