When less is not more: Limits to the evolution of metabolic dependence in spatially structured microbial communities

Summary In microbial communities, evolutionary processes can lead to loss of biosynthetic pathways, creating metabolic dependencies. The Black Queen Hypothesis suggests that such gene loss can confer a fitness advantage by reducing metabolic burden. However, how these dependencies evolve at the level of individual cells in spatially structured communities remains poorly understood. We used a combination of microfluidic single-cell imaging and mathematical modeling to examine the early fate of auxotrophic mutants within E. coli populations. We found that without external amino acids, auxotroph growth is strongly constrained by low amino acid leakage from wildtype neighbors, and further reduced when they form local clusters that drain this limited amino acid pool. A growth advantage was only observed when amino acids were added or when leakage from wildtypes exceeded a threshold. Together, our results reveal insights into determinants of mutant invasion fitness and the trade-offs between reducing metabolic costs and maintaining metabolic autonomy. Highlights Single-cell imaging reveals how auxotrophs fare in spatially structured populations Auxotrophs grow slowly due to low amino acid leakage from wildtype neighbors Mutant clustering intensifies local amino acid depletion and reduces auxotroph growth Model identifies leakage threshold required for auxotroph invasion from rare Competing Interest Statement The authors have declared no competing interest. Footnotes Abstract has been shortened. Highlights has been included. Materials and methods section has been revised according the journal requirements. Key resources table has been included. Discussion section has been revised.