Abstract
Bacteria display two primary lifestyles in their environment; existing as individual planktonic cells or forming cohesive microbial communities known as biofilms. These biofilms are ubiquitous, forming on diverse surfaces including soil, living organisms, and artificial structures. Despite the importance of biofilms, there has been limited investigation into bacterial interactions within mixed communities. Consequently, the dynamics of these communities over time, whether they become more or less competitive or cooperative are poorly understood. This study aimed to elucidate the molecular mechanisms underpinning interactions within a three-species biofilm comprised of Klebsiella pneumoniae, Pseudomonas protegens and Pseudomonas aeruginosa. Biofilms were cultivated in test tubes containing polystyrene beads and were serially passaged for six months. Single isolates derived from both mixed and mono cultures at different time points were subjected to phenotypic and genomic analyses. Overall, we observed that the capacity to form biofilms varied among species, with P. protegens consistently demonstrating greater biofilm production compared to P. aeruginosa and K. pneumoniae. P. aeruginosa produced more pyoverdine than P. protegens highlighting the former’s competitive advantage in siderophore-mediated iron acquisition. Genome sequencing of 588 single isolates collected across the six months evolution, showed that most genetic variations were related to cyclic-di-GMP genes, signaling molecules crucial for regulating bacterial behavior and physiology, including biofilm formation. Morphotypic analyses showed distinct colony morphology shifts, evolving into smaller and wrinkled colonies mostly observed in isolates from mono cultures. Interestingly, these morphotypes correlated with changes in biofilm formation and other phenotypic traits, suggesting adaptive responses to interspecies competition and cooperation. Our findings provide valuable insights into the evolutionary mechanisms underlying biofilm community dynamics and highlight the role of c-di-GMP signaling in mediating these processes. Understanding these interactions can inform strategies to manage biofilm-related infections and harness beneficial biofilms in environmental applications.