E. coli long-term evolution experiment
The E. coli long-term evolution experiment is an ongoing study in experimental evolution
Experimental evolution
In evolutionary and experimental biology, the field of experimental evolution is concerned with testing hypotheses and theories of evolution by use of controlled experiments. Evolution may be observed in the laboratory as populations adapt to new environmental conditions and/or change by such...

 led by Richard Lenski
Richard Lenski
Richard E. Lenski is an American evolutionary biologist. He is the son of sociologist Gerhard Lenski. He earned his BA from Oberlin College in 1976, and his PhD from the University of North Carolina in 1982...

 that has been tracking genetic changes in 12 initially identical populations of asexual Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

 bacteria since 24 February 1988. The populations reached the milestone of 50,000 generations .

Since the experiment's inception, Lenski and his colleagues have reported a wide array of genetic changes; some evolutionary adaptation
An adaptation in biology is a trait with a current functional role in the life history of an organism that is maintained and evolved by means of natural selection. An adaptation refers to both the current state of being adapted and to the dynamic evolutionary process that leads to the adaptation....

s 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
Citric acid
Citric acid is a weak organic acid. It is a natural preservative/conservative and is also used to add an acidic, or sour, taste to foods and soft drinks...

 in the growth media.

Experimental approach

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.

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
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

) 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
Asexual reproduction
Asexual reproduction is a mode of reproduction by which offspring arise from a single parent, and inherit the genes of that parent only, it is reproduction which does not involve meiosis, ploidy reduction, or fertilization. A more stringent definition is agamogenesis which is reproduction without...

, without bacterial conjugation
Bacterial conjugation
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells...

; this limits the study to evolution based on new mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...

s and also allows genetic marker
Genetic marker
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described as a variation that can be observed...

s to persist without spreading except by common descent
Common descent
In evolutionary biology, a group of organisms share common descent if they have a common ancestor. There is strong quantitative support for the theory that all living organisms on Earth are descended from a common ancestor....



Each of the 12 populations is kept in an incubator in Lenski's laboratory at Michigan State University
Michigan State University
Michigan State University is a public research university in East Lansing, Michigan, USA. Founded in 1855, it was the pioneer land-grant institution and served as a model for future land-grant colleges in the United States under the 1862 Morrill Act.MSU pioneered the studies of packaging,...

 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
A cryoprotectant is a substance that is used to protect biological tissue from freezing damage . Arctic and Antarctic insects, fish, amphibians and reptiles create cryoprotectants in their bodies to minimize freezing damage during cold winter periods. Insects most often use sugars or polyols as...

 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. , 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
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...

 in the E. coli genome should have occurred multiple times.

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
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde functional group.For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structurally analogous to D-glyceraldehyde.For sugars, the D/L...

). Before the beginning of the experiment Lenski prepared an Ara+ variant (a point mutation
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...

 in the ara operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...

 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
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...

, 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
Fixation (population genetics)
In population genetics, fixation is the change in a gene pool from a situation where there exist at least two variants of a particular gene to a situation where only one of the alleles remains...

 in each population, with less than 100 total point mutations (including neutral mutation
Neutral mutation
In genetics, a neutral mutation is a mutation that has no effect on fitness. In other words, it is neutral with respect to natural selection.For example, some mutations in a DNA triplet or codon do not change which amino acid is introduced: this is known as a synonymous substitution. Unless the...

s) reaching fixation in each population.
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
A citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate.-Other citric acid ions:...

 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
Citric acid cycle
The citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of chemical reactions which is used by all aerobic living organisms to generate energy through the oxidization of acetate derived from carbohydrates, fats and...

) 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
Stephen Jay Gould
Stephen Jay Gould was an American paleontologist, evolutionary biologist, and historian of science. He was also one of the most influential and widely read writers of popular science of his generation....

) "that historical contingency can have a profound and lasting impact" on the course of evolution.

Another adaptation that occurred in all these bacteria was an increase in cell size and in many cultures, a more rounded cell shape. This change was partly the result of a mutation that changed the expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 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
Fitness (biology)
Fitness is a central idea in evolutionary theory. It can be defined either with respect to a genotype or to a phenotype in a given environment...

 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.

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