Fig. 2.

Noncommutative epistasis in the evolution of aerobic citrate use in E. coli. (A) Function of the two transporters involved in the innovation of strong aerobic growth on citrate (Cit⁺⁺) in E. coli. CitT is an antiporter that exchanges extracellular citrate for internal C₄-dicarboxylates (e.g., succinate, fumarate, and malate). DctA is a carboxylic acid transporter that imports C₄-dicarboxylates from the extracellular space into the cytoplasm. (B) The two possible mutational trajectories leading to the Cit⁺⁺ trait. If the mutation leading to expression of citT (+citT) occurs first, it will transform the environment leading to cross-feeding toward the double mutant. This should not occur if the dctA overexpression mutation (+dctA) occurs first. (C and D) Simulated (C) and experimentally measured (D) fitness landscapes in the DM25 medium used in the LTEE (Materials and Methods). Experimentally obtained values are reported as mean ± SEM (n = 10).