by Eric Quandt in the lab of Jeffrey Barrick at the University of Texas at Austin described the application of a new technique called Recursive Genomewide Recombination and Sequencing (REGRES) to identify potentiating mutations among the 70 present in the Ara-3 lineage that evolved Cit+. This method used multiple rounds of a process in which F plasmid based conjugation between a 33,000 generation Cit+ clone, CZB154, and the Cit− founding clone of the LTEE to purge mutations not required for either manifestation of a weak or strong form of the Cit+ trait, which they refer to as Cit++. They found that the rnk-citT module responsible for the phenotypic switch to Cit+ was sufficient to produce a weak Cit+ phenotype in the ancestor. They also identified a mutation that had occurred in the lineage leading to CZB154 after the initial evolution of Cit+ that conferred a strong, Cit++ phenotype in the ancestor absent any mutation but the rnk-citT module. This mutation, found in the regulatory region of a gene called dctA, caused a massive increase in the expression of the DctA transporter, which functions to import C4-dicarboxylates into the cell. This increased DctA expression, they found, permitted Cit+ cells to re-uptake succinate, malate, and fumarate released into the medium by the CitT transporter during import of citrate. They identified a similar mutation in Cit++ clones in the Ara-3 population that increased DctA expression by restoring function to a gene that regulates it, dcuS, that had been deactivated in the ancestral clone. Quandt et al. concluded that the dctA mutation was not involved in potentiation, but refinement. This led them to suggest that evolution of Cit+ in the Ara-3 population might have been contingent upon a genetic background and population-specific ecology that permitted the early, weak Cit+ variants to persist in the population long enough for refining mutations to arise and render growth on citrate strong enough to provide a significant fitness benefit. Quandt and colleagues later published findings definitively identifying a mutation that did potentiate Cit+ evolution. This mutation was in the gltA gene, which encodes citrate synthase, an enzyme involved in the flow of carbon into the citric acid cycle. It had the effect of increasing citrate synthase activity, and they showed that it permitted improved growth on acetate. Moreover, with the gltA mutation, the rnk-citT module that causes the Cit+ trait has a neutral-to-slightly beneficial fitness effect, while, without