(A) Different carbon sources can be utilized by E. coli during each stage in the evolution of aerobic citrate utilization in the Lenski LTEE. Initially only glucose and acetate, a byproduct of glucose metabolism, are accessible to Cit– cells. The rudimentary Cit+ trait enabled very limited utilization of some of the abundant citrate present in the growth medium. Once this ability was refined to the Cit++ phenotype by further mutations, all of the citrate present was fully exploited. The areas of each box roughly reflect the relative amounts of each of the three carbon sources. (B) The initial gltA1 mutation potentiated the evolution of Cit+. This mutation and additional mutations in iclR and arcB all improve acetate utilization in Cit– cells by increasing flux through the TCA and glyxolyate cycles. The order of accumulation of these mutations, interspersed with many other mutations during the LTEE, is represented by arrows on the left side of the figure. These mutations may potentiate the evolution of the weak Cit+ phenotype because they create a potentiated version of central metabolism that is capable of replenishing succinate and/or other C4-dicarboxylates that must be exported in exchange for citrate import by the CitT antiporter. In accordance with this model, the addition of the CitT-activating mutation (citT) to an evolved genetic background that crucially contains the gltA1 mutation results in a rudimentary Cit+ phenotype that is neutral or slightly beneficial to fitness and thus represents a path accessible to evolution, denoted by an arrow to mark the transition as shown in left bar graph. In contrast, adding the citT mutation to the same genetic background but without the gltA1 mutation is highly deleterious to fitness, rendering this path inaccessible to evolution within the LTEE population, as shown in right bar graph. (C) Fitness effects of gltA alleles show sign epistasis with nutrient utilization niche. The gltA1 mutation that increased citrate synthase activity was beneficial to fitness when it evolved prior to the Cit+ innovation when only glucose and acetate could be utilized, as shown in the bar graph on the left. While necessary for the evolution of the rudimentary Cit+ phenotype by citT activation, the effect of gltA1 mutation is suboptimal for fitness once the dctA* mutation evolves and the citrate utilization niche can be fully exploited. Under this condition, the gltA1 mutation was deleterious to fitness and gltA2 mutations evolved that reversed the effect of the gltA1 mutation and even further reduced citrate synthase activity below the ancestral level to improve fitness in this niche, as shown in bar graph on the right. Note that relative fitness is on a different scale in each of the fitness diagrams, as all Cit++ strains are considerably more fit than any Cit– strain due to the abundant citrate in the growth medium.