Competition between Pseudomonas species constrains ecological diversification in polymicrobial biofilms

Polymicrobial biofilms are acknowledged as evolutionary hotspots; however, the influence of interspecies interactions on adaptive processes remains ambiguous. Using custom-engineered 3D-printed flow systems, biofilms were grown over an 18-day period to explore the evolutionary dynamics over time between two competing species, Pseudomonas defluvii and Pseudomonas brenneri, within communities of differing complexity, using whole-population and whole-genome sequencing methodologies. P. defluvii demonstrated significant phenotypic and genetic diversification in simple biofilms, yet this variation was reduced in complex biofilms, implying that interspecies interactions constrained its adaptation. In contrast, P. brenneri exhibited negligible evolutionary changes irrespective of the diversity present. Genomic analysis correlated the adaptation of P. defluvii with biofilm regulation and chemotaxis. The co-culture with evolved strains indicated that the variants of P. defluvii outperformed their ancestral forms, while P. brenneri remained unchanged. These results show that while the biofilm lifestyle generally fosters adaptive evolutionary processes, interspecies diversity can restrict diversification in a manner specific to each species. This study provides novel insights into the evolution of bacteria within complex biofilm environments, with implications for understanding microbial behavior in both clinical and industrial settings.