Decoding the impact of interspecies interactions on biofilm matrix components

Multispecies biofilms consist of complex communities where extracellular polymeric substances (EPS) are vital in their structure, adaptability, and function. However, characterizing the components of EPS, particularly glycans and proteins, remains a challenge due to the diverse species and their interactions within the matrix. This study examined how interactions between different species affect EPS components' production and spatial organization. We utilized a consortium of four bacterial soil isolates that have previously demonstrated various intrinsic properties in biofilm communities: Microbacterium oxydans, Paenibacillus amylolyticus, Stenotrophomonas rhizophila, and Xanthomonas retroflexus. We used fluorescence lectin-binding analysis (FLBA) to identify specific glycan components and meta-proteomics to characterize matrix proteins in mono- and multispecies biofilms. Our results revealed diverse glycan structures and compositions, including fucose and different amino sugar-containing polymers, with substantial differences between monospecies and multispecies biofilms. In isolation, M. oxydans produced galactose/N-Acetylgalactosamine network-like structures and influenced the matrix composition in multispecies biofilms. Proteomic analysis revealed flagellin proteins in Xanthomonas and Paenibacillus, particularly in multispecies biofilms. Additionally, surface-layer proteins and a unique peroxidase were found in P. amylolyticus multispecies biofilms, indicating enhanced oxidative stress resistance and structural stability under these conditions. This study highlights the crucial role of interspecies interactions in shaping biofilm matrices and the production of glycans and proteins. These findings deepen our understanding of biofilm complexity and may lead to new approaches for controlling biofilms in various environments.