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. Significance StatementCross-feeding interactions are pervasive in microbial communities, often arising from metabolite leakage into the environment. Our study focused on understanding how cross-feeding interactions shape the evolution of populations that cross-feed on these resources. Using a highly trackable cross-feeding system and linking genotypic to phenotypic changes, we showed that cross-feeding interactions reshape the evolutionary trajectories of consumer species. Notably, selection acts on flagellar genes, but their effects on function depend on the origin of resources. When resources are externally supplied, selection favors loss of flagellar motility, whereas when resources are generated through cross-feeding, selection not only maintains functional motility but also enhance it. These findings highlight that navigating the environment is essential for exploiting high-energy resources generated through cross-feeding.