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

Abstract

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. HighlightsO_LISingle-cell imaging reveals how auxotrophs fare in spatially structured populations C_LIO_LIAuxotrophs grow slowly due to low amino acid leakage from wildtype neighbors C_LIO_LIMutant clustering intensifies local amino acid depletion and reduces auxotroph growth C_LIO_LIModel identifies leakage threshold required for auxotroph invasion from rare C_LI