aerobic growth on other substances, including glucose. Most E. coli can grow anaerobically on citrate via fermentation, if a co-substrate such as glucose is available to provide reducing power. The anaerobic growth is possible due to the expression of a transmembrane citrate-succinate antiporter gene, citT, which was first identified in 1998. This gene is co-regulated with other genes involved in citrate fermentation found on the cit operon, which is turned on only when oxygen is absent. The inability to grow aerobically on citrate, referred to as a Cit− phenotype, is considered a defining characteristic of E. coli as a species, and one that has been a valuable means of differentiating E. coli from pathogenic Salmonella. Although Cit+ strains of E. coli have been isolated from environmental and agricultural samples, in every such case, the trait was found to be due to the presence of a plasmid that carries a foreign citrate transporter. A single, spontaneous Cit+ mutant of E. coli was reported by Hall in 1982. This mutant had been isolated during prolonged selection for growth on another novel substance in a growth broth that also contained citrate. Hall's genetic analysis indicated the underlying mutation was complex, but he was ultimately unable to identify the precise changes or genes involved, leading him to hypothesize activation of a cryptic transporter gene. The genome regions to which Hall was able to narrow down the locations of the changes do not correspond to the known location of the citT gene identified 16 years later, nor did the physiological characteristics in transport assays of Hall's Cit+ mutants match those to be expected for aerobic expression of the CitT transporter. Cit+ evolves in the LTEE In 2008, Lenski's team, led by Zachary D. Blount, reported that the ability to grow aerobically on citrate had evolved in one population. Around generation 33,127, a dramatic increase in turbidity was observed in the population designated Ara-3. They found that the population contained clones that were able to grow aerobically on citrate (Cit+). This metabolic capacity permitted the population to grow several-fold larger than it had previously, due to the large amount of citrate present in the medium. Examination of frozen fossil samples of the populations showed that Cit+ clones could be isolated as early as 31,500 generations. The Cit+ variants in the population were found to possess a number of genetic markers unique to the Ara-3 population; this