Prophage induction states drive structural and synergistic outcomes in marine bacterial biofilms

Microbes commonly live in biofilms, dense spatially structured assemblages where temperate phages can influence interactions amongst closely related strains. Yet, how these phage-host interactions impact biofilm architecture and ecological dynamics remains poorly understood, particularly in situations where antagonistic strains coexist in structured communities. In the marine Roseobacteraceae Sulfitobacter pontiacus, two genetically similar heteroimmune prophages, (termed A and D) establish lysogeny in their host. Depending on the host plasmid genotype, prophages are fixed in either high or low induction states, which consequently shape both biofilm structure and competitive outcomes. Using confocal scanning laser microscopy of fluorescently labeled lysogens, we investigated how prophage induction and phage genotype of phage-host coalitions influence monoculture and co-culture (competitive) S. pontiacus biofilms grown at a fluid-solid interface. Compared to their low induction counterparts, high-induction strains formed thicker and more voluminous monoculture biofilms, which primarily result from vertical protrusions arising from a basal monolayer. In co-culture experiments, mixing strains with reciprocal prophage genotypes that can mutually kill each other through phage-mediated allelopathy, we observed unexpected coexistence and synergistic biofilm enhancement rather than competitive exclusion. Reactive oxygen species (ROS) levels were significantly higher in co-cultures than monocultures and correlated with local cell density, suggesting that phage-mediated interactions generate hotspots of oxidative stress, which is expected to further promote prophage induction, creating a positive feedback loop that amplifies cell lysis and release of matrix components. These results suggest temperate phages can paradoxically stabilize and promote biofilm growth via mechanisms that combine antagonistic killing with cooperative matrix production, revealing how phage-mediated interactions can shape microbial community architecture in spatially structured environments.