Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability (Cit⁺ ) to utilize abundant citrate in the growth medium after ~31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit⁺ variants achieve a much higher population density in this environment. All Cit⁺ individuals share a mutation that activates aerobic expression of the citT citrate:C₄-dicarboxylate antiporter, but this mutation confers only an extremely weak Cit⁺ phenotype on its own. To determine which of the other >70 mutations in early Cit⁺ clones were needed to take full advantage of citrate, we developed a Recursive Genome-Wide Recombination and Sequencing (REGRES) method and performed genetic backcrosses to purge mutations not required for Cit⁺ from an evolved strain. We discovered a mutation that increased expression of the dctA C₄-dicarboxylate transporter greatly enhanced the Cit⁺ phenotype after it evolved, implicating the intracellular supply of succinate or other C₄-dicarboxylates to be a critical factor for the expression of the phenotype. The activity level of citrate synthase (CS), encoded by the gltA gene, was also found to be important for Cit⁺. A mutation to gltA (gltA1) occurred before the evolution of Cit⁺ and led to an increase in CS activity by diminishing allosteric inhibition by NADH. This mutation was found to be deleterious for high-level citrate utilization, a situation that was remedied shortly after the evolution of Cit⁺ by the evolution of compensatory mutations to gltA which decreased CS activity. We speculate that the gltA1 mutation may have been important to 'potentiate' the evolution of a weak Cit⁺ phenotype by increasing succinate production via an upregulated glyoxylate pathway but that as cells became able to import succinate by virtue of the dctA mutation that this pathway became maladaptive, prompting this evolutionary reversal. Overall, our characterization of this metabolic innovation highlights the degree to which interactions between alleles shape the evolution of complex traits and emphasizes the need for novel whole-genome methods to explore such relationships.

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