Reciprocal cross-feeding between bacteria can limit the emergence of metabolic dependencies

Cross-feeding is prevalent in microbial communities. Through time, cross-feeding is thought to enrich for loss-of-function mutations, thereby creating or reinforcing dependencies between community members. However, few studies have compared how cross-feeding affects the evolutionary trajectory of partners compared to monoculture conditions. Here, we compared mutations that were differentially enriched in bacterial monocultures versus cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli in an obligate cross-feeding relationship based on the exchange of nitrogen and carbon for 650-800 generations. Opposite trends for the number of differentially enriched mutations were observed for each species; R. palustris accumulated more unique mutations in monoculture, whereas E. coli accumulated more unique mutations in co-culture. Contrary to expectations, the emergence of additional dependencies was observed in monoculture, but not in coculture, even though additional layers of cross-feeding involving iron and adenine were present in coculture. We reasoned that iron and adenine cross-feeding occurred at levels sufficient to repress gene expression in the recipient, thereby promoting gene retention by lowering gene cost. We also observed that E. coli acquired mutations in coculture that were suggestive of enhanced adenine uptake, whereas R. palustris gained the ability to oxidize H₂, but only in monoculture. Thus, the influence of cross-feeding on evolutionary trajectories can vary with organisms and conditions, and there are situations where cross-feeding can limit, rather than promote, emergent dependencies.IMPORTANCEBacteria commonly engage in cross-feeding, where nutrients are transferred between neighbors. Cross-feeding is thought to alleviate energy expenditures for genes whose role can be met by cross-fed nutrients, leading to eventual gene loss. However, few examples have been documented, especially in comparison to monocultures that lack a cross-feeding partner. We grew cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli alongside corresponding monocultures for 650-800 generations. While coculture conditions required obligate exchange of nitrogen and carbon, additional cross-feeding of adenine and iron likely occurred. Contrary to expectations, dependencies for iron and unknown compounds emerged in monocultures, but expected iron and adenine dependencies were not observed in cocultures. Low expression of iron scavenging and adenine synthesis genes in cocultures suggested that cross-feeding repressed expression, thereby lowering gene cost. Thus, although cross-feeding can sometimes make costly genes dispensable, there are also cases where cross-feeding lowers gene cost, thereby promoting gene retention.