After ~33,000 generations, or 16 years into Dr. Lenski’s experiment, population #9 suddenly became much larger. Dr. Lenski’s students discovered that a lineage in this population evolved the ability to grow on the citrate in the bacteria’s nutrient broth at about the 31,000 generation time point.
The Use of Citrate
E. coli is normally unable to grow on citrate when oxygen is present. However, it has a citric acid cycle, meaning that it can metabolize citrate during aerobic growth on other substances. E. coli therefore has the cellular machinery to grow on citrate if it can transport it into the cell. E. coli can grow on citrate when no oxygen is present through anaerobic processes. E. coli is able to bring citrate into the cell under these conditions because it has a gene called citT that encodes a transmembrane citrate-succinate antiporter. However, citT is part of an operon containing genes needed for citrate fermentation that is only turned on in the absence of oxygen.
CitT is a transmembrane antiporter than can bring a broad array of di- and tri-carboxylic acids into the cell in exchange for other di- and tri-carboxylic acids. This exchange is a form of secondary active transport in which the energy for importing one substrate is provided by the export of the other substrate down an electrochemical gradient from high concentration to low concentration, with no need for ATP to drive the process. CitT principally functions as a citrate-succinate antiporter, which makes it ideal for citrate processing, in which succinate is an end product. In the Cit+ lineage in LTEE population #9, a complex mutation allows the cells to express the CitT transporter in the presence of oxygen (See the molecular genetics section) and thereby access the citrate resource. When citrate is pumped into a Cit+ cell, it enters the citrate acid cycle directly, as opposed to citrate synthesized from oxaloacetate and acetyl-CoA during glucose metabolism. The citrate is then converted to succinate through the citric acid cycle’s reactions, producing energy in the process. The succinate is then pumped out into the medium in exchange for more citrate.