Exchange of metabolic pathway intermediates as the origin for metabolite cross-feeding interactions in E. coli auxotrophic co-cultures

Abstract

A large fraction of microbes in natural environments are auxotrophs that rely on metabolite exchange for growth. The Black Queen hypothesis and related frameworks explain gene loss and reliance on shared metabolites. Yet it is not fully understood how metabolite cross-feeding emerges and gives rise to experimentally observable community performance. Here, we propose that exchange of biosynthetic pathway intermediates provides a mechanistic route for the emergence of metabolite cross-feeding. Building on prior work demonstrating intermediate exchange in E. coli auxotrophs, we investigated co-cultures between a methionine auxotroph ({Delta}metA) and a panel of histidine auxotrophs. Among these, only {Delta}hisD exhibited efficient co-culture growth with {Delta}metA, suggesting a unique role for the final step of histidine biosynthesis, which converts histidinol to histidine. We found that {Delta}metA-{Delta}hisD co-cultures accumulated high levels of extracellular histidinol (>200 M), suggesting a mechanism for metabolite exchange. Spent media from these co-cultures supported growth of all histidine auxotrophs except {Delta}hisD, confirming that histidinol, rather than histidine, was the dominant exchanged metabolite. Furthermore, we observed that {Delta}metA efficiently converted histidinol to histidine only under methionine-limited conditions, suggesting that nutrient limitation promoted overflow of converted histidine to the extracellular environment. To quantitatively assess whether histidinol exchange could account for the observed co-culture behavior, we constructed a mechanistic model that links extracellular histidinol dynamics to co-culture growth through independently measured conversion capacity of the {Delta}metA strain. Rather than fitting growth empirically, we used experimentally derived parameters to predict co-culture growth from the measured histidinol-to-histidine conversion flux. The model accurately recapitulated the timing and magnitude of growth and population dynamics, supporting the idea that accumulation of histidinol enabled the conversion-driven supply of histidine that sustained {Delta}hisD, while coupling growth between the two strains. Taken together, these results suggest that metabolite cross-feeding in syntrophic co-cultures can emerge from the accumulation of pathway intermediates and their conditional conversion under nutrient limitation, providing a plausible mechanism for the rapid establishment of mutualistic interactions.