Microbial cross-feeding interactions reshape evolutionary trajectories of consumers by preserving motility

Cross-feeding interactions, in which a producer species release by-products that serve as resources for a consumer species, play an important role in shaping microbial community diversity. Producers create opportunities for consumers by supplying high-energy resources that are often scarce in the environment. However, they also exert strong top-down effects by releasing metabolites in pulses and generating spatial gradients of resource availability. How these spatiotemporal constraints shape consumer evolution remains poorly understood. To address this question, we used a two-species cross-feeding system in which Acinetobacter johnsonii excretes benzoate (a by-product of benzyl alcohol oxidation) into the external environment where it is consumed by Pseudomonas putida . To assess how the origin of benzoate (externally supplied or produced by cross-feeding) shapes consumer evolution, we evolved P. putida for 200 generations in monoculture or in co-culture with A. johnsonii . Populations evolved in monoculture exhibited improved growth relative to the ancestor, whereas populations evolved under cross-feeding showed little to no growth improvement.

Whole-genome sequencing revealed pervasive loss-of-function mutations in flagellar genes among populations evolved in monoculture, but not under cross-feeding conditions. High-throughput imaging assays showed that populations evolved under cross-feeding not only maintained but also enhanced functional motility. Competition experiments with single mutants revealed context-dependent fitness effects: loss-of-function mutations were highly beneficial when benzoate was externally supplied but deleterious when benzoate was supplied by A. johnsonii , highlighting the importance of motility in cross-feeding interactions. Together, our results show that resource origin fundamentally reshapes selective pressures and alters evolutionary outcomes in microbial communities.