Functional and ecological drivers of bacterial interactions in glacier-fed stream biofilms.

Biofilms are the dominant bacterial lifestyle, consisting of matrix-embedded, spatially structured communities where bacteria closely interact and coordinate their behaviors. Despite their ecological significance, the interplay among bacterial taxa that shapes biofilm community dynamics remains poorly characterized. We assessed the nature and drivers of interactions among bacterial strains isolated from a glacier-fed stream biofilm, using phenotyping, genomics, and metabolic fingerprinting. Biofilm bacterial coexistence was frequent (37% of pairs) even among strains with overlapping resource use and close phylogenetic relatedness. Positive interactions were evidenced through metabolic cross-feeding, synergy in biofilm formation, and interspecific biofilm induction. In competitive interactions, where one strain outcompeted the other, the dominant bacteria exhibited oligotrophic traits, such as higher carbon use efficiency, substrate specialization, high-yield strategies, and the production of antagonistic molecules. Our integrated approach demonstrates that efficient growth traits confer a competitive advantage, while widespread metabolic cooperation can facilitate bacterial coexistence. These findings provide critical insights into the forces shaping bacterial interactions and community assembly in stream biofilms.IMPORTANCEBacterial isolates have been extensively used across many systems to investigate how their interactions and traits shape coexistence and competition patterns. However, stream biofilm bacteria, despite forming the foundation of fluvial microbial ecosystems, have rarely been studied beyond their taxonomic composition, although deciphering their interactions is key to understanding ecosystem functioning. Here, we leveraged biofilm isolates from a glacier-fed stream to examine both competitive and positive interactions in co-culture, revealing the nature and key bacterial traits that mediate these interactions. By linking co-culture outcomes to microbial traits, this study uncovers the drivers of bacterial interactions in stream biofilm communities.