Type IV pilus shapes a "bubble-jet" pattern opposing spatial intermixing of two interacting bacterial populations

Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be an important determinant of community-level properties. The best-studied characteristics of spatial patterns is spatial intermixing of different populations. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (referred to here as a bubble-jet pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding for type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments. ImportanceWhen growing on surfaces, multi-species microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, such as enabling otherwise impermissible metabolic functions to occur, as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganism, but its role on pattern formation within microbial communities is unclear. Here we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation, and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy to engineer high-performance synthetic microbial communities.