E. coli long-term evolution experiment

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The 12 evolving E. coli populations on June 25, 2008

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 and 60,000 in April 2014.[2]

Zachary Blount and Richard Lenski in 2015

Since the experiment's inception in 1988, Lenski and his colleagues have reported a wide array of genetic changes. Some changes occurred in all 12 populations and others have only appeared in one or a few populations. For example, all 12 populations experienced improvement in fitness that decelerated over time and some of populations evolved detrimental effects such as defects in DNA repair, causing mutator phenotypes. One of the significant adaptions occurred in one strain of E. coli. In general, this bacteria is known for not being able to use citrate in an aerobic environment as an energy source, even though it could use citrate under anaerobic conditions because it already has the machinery to process citrate.[3] This strain, though ancestrally unable to do so initially, was able to transport citrate for use as an energy source after a duplication mutation that was involved in the gene for the citrate transporter protein used in anaerobic growth. Even though all the ancestors already had a complete citric acid cycle, and thus could metabolize citrate internally for energy during aerobic growth, none of the 12 populations had a transporter protein for citrate since the beginning, which was the only barrier to being able to use citrate for energy in oxygen-rich conditions. Earlier independent studies had already reported E. coli strains from agricultural or clinical settings that already had the ability to use citrate under aerobic conditions.[4]

A genomic study was done to investigate the history of the adaption using clones to isolate the number of mutations needed to develop the trait. It concluded that multiple mutations (at least two or more) such as duplication mutations were needed to allow the transport of citrate for use in energy. For the trait to develop and stick in a population, it needed multiple mutations at three main phases: potentiation (makes a trait possible), actualization (makes the trait manifest), and refinement (makes it effective).[5]

Background on the growth of E. coli using citrate[edit]

E. coli is normally unable to grow on citrate when oxygen is present (aerobic conditions).[3] However, since E. coli does have a citric acid cycle, it is not entirely indifferent to citrate even when oxygen is present because, for instance, E. coli can metabolize citrate provided certain available sources of carbon and energy are simultaneously present in the medium such as glucose or fructose.[3][4][6] Normally, E. coli has the cellular machinery to grow on citrate under anaerobic conditions since it is able to bring citrate into the cell when no oxygen is present because of a gene called citT that encodes a transmembrane citrate-succinate antiporter.[3] However, citT is part of an operon containing genes needed for citrate fermentation that is only turned on in the absence of oxygen.[3]

Experimental approach[edit]

The long-term evolution experiment was intended to provide experimental evidence for several of the central questions 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.[7]

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.[7]


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. Under these conditions, each population experiences 6.64 generations, or doublings, each day. Large, representative samples of each population are frozen with glycerol as a cryoprotectant at 500-generation (75 day) intervals. The bacteria in these samples remain viable, and can be revived at any time. This collection of samples is referred to as the "frozen fossil record", and provides a history of the evolution of each population through the entire experiment. The populations are also regularly screened for changes in mean fitness, and supplemental experiments are regularly performed to study interesting developments in the populations.[8] As of October 2012, the E. coli populations have been under study for over 56,000 generations, and are thought to have undergone enough spontaneous mutations that every possible single point mutation in the E. coli genome has occurred multiple times.[4]

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, rm, 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.


Growth in cell size of bacteria in the Lenski experiment

In the early years of the experiment, several common evolutionary developments were shared by the populations. The mean fitness of each population, as measured against the ancestor strain, increased, rapidly at first, but leveled 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, four 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 within this time frame, only 10 to 20 beneficial mutations achieved fixation in each population, with fewer than 100 total point mutations (including neutral mutations) reaching fixation in each population.[7]

The population designated Ara-3 (center) is more turbid because that population evolved to use the citrate present in the growth medium.

Evolution of aerobic citrate usage in one population[edit]

In 2008, Lenski and his collaborators reported an adaptation that occurred in the population called Ara-3: the bacteria evolved the ability to use citrate under the oxygen-rich conditions via a citrate transporter. Wild-type E. coli generally cannot use citrate when oxygen is present due to the inability during aerobic metabolism to produce an appropriate transporter protein that can bring citrate into the cell, where it could be metabolized via the citric acid cycle. The consequent lack of growth on citrate under oxic conditions, referred to as a Cit phenotype, is considered a defining characteristic of E. coli that has been a valuable means of differentiating E. coli from pathogenic Salmonella. However, in previous literature there had already been research from clinical and agricultural settings reporting of strands of E. coli that were able to use citrate as an energy source as well and had acquired the missing citrate transporter presumably from other species. Furthermore, E. coli in general is not wholly indifferent to citrate since it has a citric acid cycle which already can metabolize citrate along with other substrates and it can ferment citrate elsewhere.[3][4]

Around generation 33,127, they saw a dramatically expanded population-size in one of the samples indicating that this population could grow in a medium with citrate. This led to the discovery that a citrate-using variant of E. coli (Cit+) 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 re-evolve in a subset of genetically pure clones from earlier time points in the population's history. Such re-evolution of citrate use was never observed in clones isolated from before generation 20,000. Even in those clones that were able to re-evolve citrate use, the function showed a rate of occurrence on the order of one occurrence per trillion cell divisions.[4]

The authors interpret these results as indicating that the evolution of citrate use in this one population depended on one or more earlier, possibly nonadaptive "potentiating" mutations that increased the rate of mutation to an accessible level. The data suggests that citrate usage required at least two mutations subsequent to these "potentiating" mutations. More generally, the authors suggest 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.[4]

In 2012, Lenski and his team reported the results of a genomic analysis of the Cit+ trait that shed light on the genetic basis and evolutionary history of the trait. The researchers had sequenced the entire genomes of twenty-nine clones isolated from various time points in the Ara-3 population's history. They used these sequences to reconstruct the phylogenetic history of the population, which showed that the population had diversified into three clades by 20,000 generations. The Cit+ variants had evolved in one of these, which they called Clade 3. Clones that had been found to be potentiated in earlier research were distributed among all three clades, but were over-represented in Clade 3. This led the researchers to conclude that there had been at least two potentiating mutations involved in Cit+ evolution.[5]

The researchers also found that all Cit+ clones had duplication mutations of a 2933 base pair segment that were involved in the gene for the citrate transporter protein used in anaerobic growth on citrate, citT. The duplication is tandem and resulted in two copies that were head-to-tail with respect to each other. This duplication immediately conferred the Cit+ trait by altering the regulation in which the normally silent citT gene is placed under the control of a promoter for an adjacent gene called rnk. The new promoter activated the expression of the citrate transporter when oxygen was present, and thereby enabled aerobic growth on citrate.[5]

Movement of this rnk-citT module into the genome of a potentiated Cit clone was shown to be sufficient to produce a Cit+ phenotype. However, the initial Cit+ phenotype conferred by the duplication was very weak, and only granted a ~1% fitness benefit. The researchers found that the number of copies of the rnk-citT module had to be increased to strengthen the Cit+ trait sufficiently to permit the bacteria to grow well on the citrate. Further mutations after the Cit+ bacteria became dominant in the population continued to accumulate improved growth on citrate. The researchers concluded that the evolution of the Cit+ trait suggests that new traits evolve through three stages: potentiation (making the trait possible); actualization, (making the trait manifest); and refinement (making the trait effective).[5]

Evolution of increased cell size in all twelve populations[edit]

All twelve of the experimental populations show an increase in cell size, and in many of the populations, a more rounded cell shape.[9] 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 outcompete 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.[9]

Continued increase in fitness[edit]

In 2013, the team reported that after 50,000 generations in a challenging environment, the bacteria were continuing to improve their abilities. Comparing the behaviour of all strains with samples from the 40,000 batch, the mean fitness appears to be increasing without bound.[10]

A paper published in December 2015 detailing the results of more than 1100 new competitive fitness assays after a further 10,000 generations shows that the increase of an evolving population's mean fitness is best explained by a power law model, rather than a hyperbolic model. In a power law model the rate of fitness gain declines over time, but there is no upper limit, whereas the hyperbolic model implies a hard limit. The results suggest that both adaptation and divergence can increase for a long time, perhaps indefinitely, even in a constant environment.[11][12]

See also[edit]


  1. ^ a b Lenski, Richard E. (2000). "Source of founding strain". Richard E. Lenski Homepage. Michigan State University. Retrieved 2008-06-18. 
  2. ^ @LayneCameron1 (May 15, 2014). "Window to evolution. #MSU lab that bottled evolution has produced 60K generations of bacteria!". Twitter. 
  3. ^ a b c d e f "Cell Biology: The Use of Citrate". EVO-ED. University of Michigan. 
  4. ^ a b c d e f Blount, Zachary D.; Borland, Christina Z.; Lenski, Richard E. (2008). "Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli". Proceedings of the National Academy of Sciences 105 (23): 7899–906. Bibcode:2008PNAS..105.7899B. doi:10.1073/pnas.0803151105. JSTOR 25462703. PMC 2430337. PMID 18524956. 
  5. ^ a b c d Blount ZD, Barrick JE, Davidson CJ, Lenski RE (2012-09-27). "Genomic analysis of a key innovation in an experimental Escherichia coli population". Nature 489 (7417): 513–518. Bibcode:2012Natur.489..513B. doi:10.1038/nature11514. PMC 3461117. PMID 22992527. 
  6. ^ Lara, F.J.S; Stokes, J.L. (1952). "Oxidation of citrate by Escherichia coli". Journal of Bacteriology 63 (3): 415–420. PMC 169284. PMID 14927574. 
  7. ^ a b c Lenski, Richard E. (2003). Janick, Jules, ed. "Phenotypic and Genomic Evolution during a 20,000-Generation Experiment with the Bacterium Escherichia coli". Plant Breeding Reviews (New York: Wiley) 24 (2): 225–65. doi:10.1002/9780470650288.ch8. ISBN 978-0-471-46892-9. 
  8. ^ Lenski, Richard E. (2000). "Overview of the E. coli long-term evolution experiment". Richard E. Lenski Homepage. Michigan State University. Retrieved 2008-06-18. 
  9. ^ a b Philippe, Nadège; Pelosi, Ludovic; Lenski, Richard E.; Schneider, Dominique (2008). "Evolution of Penicillin-Binding Protein 2 Concentration and Cell Shape during a Long-Term Experiment with Escherichia coli". Journal of Bacteriology 191 (3): 909–21. doi:10.1128/JB.01419-08. PMC 2632098. PMID 19047356. 
  10. ^ Wiser, Michael J.; Ribeck, Noah; Lenski, Richard E. (13 December 2013). "Long-Term Dynamics of Adaptation in Asexual Populations". Science 342 (6164): 1364–1367. Bibcode:2013Sci...342.1364W. doi:10.1126/science.1243357. 
  11. ^ Scharping, Nathaniel. "Could Evolution Ever Yield a ‘Perfect’ Organism?". Discover Magazine. Archived from the original on 18 December 2015. Retrieved 18 December 2015. 
  12. ^ Lenski, Richard E; et al. "Sustained fitness gains and variability in fitness trajectories in the long-term evolution experiment with Escherichia coli". Proceedings B. The Royal Society. Archived from the original on 18 December 2015. Retrieved 18 December 2015. 

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