ABSTRACT
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. For instance, mature biofilms of Bacillus subtilis contain cells in an ‘ON’ state, expressing extracellular matrix genes, as well as cells in an ‘OFF’ state. 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. In addition, the influence of cheats on phenotypic heterogeneity pattern within matrix-producers, was never examined. Here, we describe the long-term dynamics between EPS producers (cooperators) and non-producers (cheaters) in B. subtilis biofilms and track changes in phenotypic heterogeneity of matrix production within the populations of co-operators. We discovered that cheater-mediated evolution in pellicles leads to a transient shift in phenotypic heterogeneity pattern of co-operators, namely an increased number of eps expressing cells as depicted by hyper ON phenotype. Although hyper ON strategy seems adaptive in presence of cheats, it is soon substituted by hyper OFF phenotype and/or soon after by population collapse. This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.
SIGNIFICANCE STATEMENT Microbial biofilms are significant in medical, environmental and industrial settings. Biofilm control strategies have been proven to be challenging due to their increased resistance to antimicrobials. Here, we employ a cheater-mediated evolution study in Bacillus subtilis pellicles to understand in long-term scale how biofilms’ social behavior evolves as triggered by stress. We show that evolution of matrix-producing cells (cooperators) in the presence of non-producers (cheaters) leads to a cheating strategy that allows cheaters to benefit from cooperators, that subsequently result to population tragedy. However, cooperators can also adapt and evade exploitation via an anti-cheating system that involves shift in phenotypic heterogeneity related to biofilm matrix expression. This study highlights biofilm adaptation and stress response mechanisms within the context of evolution.