The LTEE populations are transferred daily into fresh medium, and the turbidity of each is checked visually at that time. Owing to the low concentration of glucose in DM25 medium, the cultures are only slightly turbid when transferred. Occasional contaminants that grow on citrate have been seen over the 20 years of this experiment. These contaminated cultures reach much higher turbidity owing to the high concentration of citrate in the medium, which allows the contaminants to reach high density. (When contamination occurs, the affected population is restarted from the latest frozen sample.) After ?33,127 generations, one population, designated Ara-3, displayed significantly elevated turbidity that continued to rise for several days (Fig. 1). A number of Cit+ clones were isolated from the population and checked for phenotypic markers characteristic of the ancestral E. coli strain used to start the LTEE: all were Ara-, T5-sensitive, and T6-resistant, as expected (2). DNA sequencing also showed that Cit+ clones have the same mutations in the pykF and nadR genes as do clones from earlier generations of the Ara-3 population, and each of these mutations distinguishes this population from all of the others (30). Therefore, the Cit+ variant arose within the LTEE and is not a contaminant.
The evolved Cit+ variant grows to high density in DM0 (a citrate-only medium), produces vigorous colonies on minimal citrate (MC) agar plates, and causes a positive color change on Simmon's citrate agar, all of which indicate that it can use citrate as a sole carbon source. In DM25, Cit+ cells undergo a period of rapid growth on glucose that is followed by slower growth on citrate (Fig. 2). Also, growth on citrate is inhibited by the citrate analog 5-fluorocitrate (data not shown), as was observed for the one previously reported Cit+ mutant of E. coli (42, 43).
Cit+ clones could be readily isolated from the frozen sample of population Ara-3 taken at generation 33,000. To estimate the time of origin of the Cit+ trait, we screened 1,280 clones randomly chosen from generations 30,000, 30,500, 31,000, 31,500, 32,000, 32,500, and 33,000 for the capacity to produce a positive reaction on Christensen's citrate agar, which provides a sensitive means to detect even weakly citrate-using cells. No Cit+ cells were found in the samples taken at 30,000, 30,500, or 31,000 generations. Cit+ cells constituted ?0.5% of the population at generation 31,500, then 15% and 19% in the next two samples, but only ?1.1% at generation 33,000. It appears that the first Cit+ variant emerged between 31,000 and 31,500 generations, although we cannot exclude an earlier origin. The precipitous decline in the frequency of Cit+ cells just before the massive population expansion suggests clonal interference (47), whereby the Cit- subpopulation produced a beneficial mutant that out-competed the emerging Cit+ subpopulation until the latter evolved some other beneficial mutation that finally ensured its persistence. The hypothesis of clonal interference implies that the early Cit+ cells were very poor at using citrate, such that a mutation that improved competition for glucose could have provided a greater advantage than did marginal exploitation of the unused citrate.
Indeed, the Cit+ clones isolated from generations 32,500 and earlier grow much more slowly on citrate than those from 33,000 generations and later. After depleting the glucose in DM25, the earliest Cit+ clones grow almost imperceptibly, if at all, for many hours before they begin efficiently using the citrate (data not shown), whereas later Cit+ clones switch to growth on citrate almost immediately (Fig. 2). Thus, the population expansion between generations 33,000 and 33,500 (Fig. 1) was triggered by one or more mutations that improved citrate utilization, rather than by the growth of the original Cit+ mutant. This finding also raises the question of whether weak Cit+ mutants might exist in any other LTEE population. We therefore screened the other 11 populations, in most cases by using samples from generation 41,500. None of 220 cultures inoculated with heterogeneous population samples grew in glucose-free DM0, nor did any of >3,500 clones show a positive reaction on Christensen's citrate agar.