eLife 10.7554/eLife.09696

Credit: eLife

In 1988, Lenski and colleagues began a long-term experimental evolution project in which E. coli were continuously grown and samples collected every ∼500 generations as a platform to explore evolutionary principles. After 31,500 generations of growth on glucose in the presence of citrate, which E. coli cannot typically metabolize under aerobic conditions, a citrate-utilizing population emerged; this population, called Cit+, was defined by duplication of the citT citrate:succinate antiporter gene and was followed by a stronger phenotype, Cit++, based in part on activated expression of the dctA symporter gene. However, it was unclear what background changes were needed to facilitate the emergence of the Cit+ and Cit++ populations. Quandt et al. now explore the role of the citrate synthase gene, gltA, in this process. In particular, the authors identified a gltA1 mutation affecting citrate synthase prior to the evolution of Cit+; in Cit++ clones, additional gltA2 mutations were observed. Genetic reconstruction confirmed that gltA2 mutations were needed to support the Cit++ phenotype. In contrast, other strain combinations showed that gltA2 mutations when citrate cannot be used as a carbon source, the gltA1 mutation in the absence of a compensating gltA2 mutation in a Cit++ background, and the duplication of citT in the absence of the gltA1 mutation were each deleterious for growth. Metabolic modeling suggested that growth on glucose would be constrained by low citrate synthase flux and that high levels of citrate synthase would improve growth on acetate (a byproduct of glucose metabolism), whereas growth on citrate would be optimal with no or low flux. Accordingly, the gltA1 mutation likely provided a competitive advantage to the pre-Cit+ cells, while the gltA2 mutations reversed the effect of the gltA1 mutations by decreasing the amount of mRNA, reintroducing lost inhibitory mechanisms or directly reducing enzyme function. These 'metabolic epochs' provide a unique glimpse into the development of new functions.