Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B.  subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.