Decoding the impact of interspecies interactions on biofilm matrix components

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