The E. coli long-term evolution experiment is an ongoing study in experimental evolution led by Richard Lenski that has been tracking genetic changes in 12 initially identical populations of asexual Escherichia coli bacteria since 24 February 1988.[1] The populations reached the milestone of 50,000 generations in February 2010[update].
Since the experiment's inception, Lenski and his colleagues have reported a wide array of genetic changes; some evolutionary adaptations have occurred in all 12 populations, while others have only appeared in one or a few populations. One particularly striking adaption was the evolution of a strain of E. coli that was able to grow on citric acid in the growth media.[2]
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The long-term evolution experiment was intended to provide experimental evidence for several of the central problems of evolutionary biology: how rates of evolution vary over time; the extent to which evolutionary changes are repeatable in separate populations with identical environments; and the relationship between evolution at the phenotypic and genomic levels.[3]
The use of E. coli as the experimental organism has allowed many generations and large populations to be studied in a relatively short period of time, and has made experimental procedures (refined over decades of E. coli use in molecular biology) fairly simple. The bacteria can also be frozen and preserved, creating what Lenski has described as a "frozen fossil record" that can be revived at any time (and can be used to restart recent populations in cases of contamination or other disruption of the experiment). Lenski chose an E. coli strain that reproduces only asexually, without bacterial conjugation; this limits the study to evolution based on new mutations and also allows genetic markers to persist without spreading except by common descent.[3]
Each of the 12 populations is kept in an incubator in Lenski's laboratory at Michigan State University in a minimal growth medium. Each day, 1% of each population is transferred to a flask of fresh growth medium. Large, representative samples of each population are frozen with glycerol as a cryoprotectant at 500-generation (75 day) intervals. The populations are also regularly screened for changes in mean fitness, and supplemental experiments are regularly performed to study interesting developments in the populations.[4] As of February 2010[update], the E. coli populations have been under study for over 50,000 generations, and are thought to have undergone enough spontaneous mutations that every possible single point mutation in the E. coli genome should have occurred multiple times.[2]
The initial strain of E. coli for Lenski's long-term evolution experiment came from "strain Bc251", as described in a 1966 paper by Seymour Lederberg, via Bruce Levin (who used it in a bacterial ecology experiment in 1972). The defining genetics traits of this strain were: T6r, Strr, r−m−, Ara− (unable to grow on arabinose).[1] Before the beginning of the experiment Lenski prepared an Ara+ variant (a point mutation in the ara operon that enables growth on arabinose) of the strain; the initial populations consisted of 6 Ara− colonies and 6 Ara+ colonies, which allowed the two sets of strains to be differentiated and tested for fitness against each other. Unique genetic markers have since evolved to allow identification of each strain.
In the early years of the experiment, there were several common evolutionary developments shared by the populations. The mean fitness of each population, as measured against the ancestor strain, increased—rapidly at first, but leveling off after close to 20,000 generations (at which point they grew about 70% faster than the ancestor strain). All populations evolved larger cell volumes and lower maximum population densities, and all became specialized for living on glucose (with declines in fitness relative to the ancestor strain when grown in dissimilar nutrients). Of the 12 populations, 4 developed defects in their ability to repair DNA, greatly increasing the rate of additional mutations in those strains. Although the bacteria in each population are thought to have generated hundreds of millions of mutations over the first 20,000 generations, Lenski has estimated that only 10 to 20 beneficial mutations achieved fixation in each population, with less than 100 total point mutations (including neutral mutations) reaching fixation in each population.[3]
In 2008, Lenski and his collaborators reported on a particularly important adaptation that occurred in one of the twelve populations: the bacteria evolved the ability to utilize citrate as a source of energy. Wild type E. coli cannot transport citrate across the cell membrane to the cell interior (where it could be incorporated into the citric acid cycle) when oxygen is present. The consequent lack of growth on citrate under oxic conditions is considered a defining characteristic of the species that has been a valuable means of differentiating E. coli from pathogenic Salmonella. Around generation 33,127, the experimenters noticed a dramatically expanded population-size in one of the samples; they found that there were clones in this population that could grow on the citrate included in the growth medium to permit iron acquisition. Examination of samples of the population frozen at earlier time points led to the discovery that a citrate-using variant had evolved in the population at some point between generations 31,000 and 31,500. They used a number of genetic markers unique to this population to exclude the possibility that the citrate-using E. coli were contaminants. They also found that the ability to use citrate could spontaneously re-evolve in populations of genetically pure clones isolated from earlier time points in the population's history. Such re-evolution of citrate utilization was never observed in clones isolated from before generation 20,000. Even in those clones that were able to re-evolve citrate utilization, the function showed a rate of occurrence on the order of once per trillion cells. The authors interpret these results as indicating that the evolution of citrate utilization in this one population depended on an earlier, perhaps non-adaptive "potentiating" mutation that had the effect of increasing the rate of mutation to citrate utilization to an accessible level (with the data they present further suggesting that citrate utilization required at least two mutations subsequent to this "potentiating" mutation). More generally the authors suggest that these results indicate (following the argument of Stephen Jay Gould) "that historical contingency can have a profound and lasting impact" on the course of evolution.[2]
Another adaptation that occurred in all these bacteria was an increase in cell size and in many cultures, a more rounded cell shape.[5] This change was partly the result of a mutation that changed the expression of a gene for a penicillin binding protein, which allowed the mutant bacteria to out-compete ancestral bacteria under the conditions in the long-term evolution experiment. However, although this mutation increased fitness under these conditions, it also increased the bacteria's sensitivity to osmotic stress and decreased their ability to survive long periods in stationary phase cultures, so the phenotype of this adaptation depends on the environment of the cells.[5]