Fine-tuning Citrate Synthase Flux Potentiates and Refines

26 27 Evolutionary innovations that enable organisms to colonize new ecological niches are rare 28 compared to gradual evolutionary changes in existing traits. We discovered that key mutations in 29 the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia 30 coli gained the ability to grow aerobically on citrate (Cit + phenotype) during the Lenski long-31 term evolution experiment. The first gltA mutation, which increases CS activity by disrupting 32 NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to 33 preserving the rudimentary Cit + trait from extinction when it first evolved. However, after Cit + 34 was refined by further mutations, this potentiating gltA mutation became deleterious to fitness. A 35 second wave of beneficial gltA mutations then evolved that reduced CS activity to below the 36 ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new 37 nutrient niche contingent on both co-opting and overcoming a history of prior adaptation.