Tag: 6. April 2016

Der experimentelle Nachweis für evolutionäre Theorien (E. Coli Langzeitstudie)

/ Christian - Alles Evolution / 37 Kommentare

Häufig wird angeführt, dass es keinen Beweis dafür geben würde, dass die Theorien der Evolution überhaupt funktionieren und dadurch tatsächlich etwas neues entstehen kann.

Allerdings gibt es ein sehr schönes Experiment, in dem ein Forscher 12 Populationen von Bakterien über mehr als 50.000 Generationen beobachtet hat und die Zwischenschritte tiefgefroren zur Verfügung stehen.

Zu der Methodik:

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, 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.

Die Bakterien hatten also eine gewisse Gelegenheit auf der immer gleichen Menge von Nährlösung zu wachsen, die Nährstoffe stellten dabei den limitierenden Faktor dar, nach dem eine Selektion innerhalb der Bakterien erfolgte. Die Bakterien, die abstarben, weil ihre Nachbarn ihnen Platz, Nährstoffe etc wegnahmen, kamen nicht in die nächste Generation. Letztendlich erfolgte damit eine Selektion auf effektive und schnelle Nährstoffnutzung und Verdrängung und Ausschalten der Konkurrenz.

Und tatsächlich kam es im Laufe des Experiments zu der Nutzung einer vollkommen neuen Nährstoffquelle, also einer positiven Selektion:

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

Evolution of aerobic citrate usage in one population
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. coligenerally 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 pathogenicSalmonella. 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] Much of this work was conducted by then graduate student Zachary Blount.

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.

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.

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.

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]

Die Zitronensäure, die von diesen entsprechend gezüchteten Bakterien genutzt werden kann, was – wenn ich es richtig verstehe – einfach der eigentlich als Nährstoffquelle vorgesehenen Glukose in der Verarbeitung beigemischt. Sie wurde durch die Mutation als zusätzlich Nährstoffquelle nutzbar.

Es wurden, wenn ich andere Berichte richtig im Kopf habe, sogar die letzten Schritte wiederholt. Man taute einige der „Bakterienfossilien“ auf und in den Vorgängerversionen in denen einige kritische Mutation bereits aufgetreten war, kam es erneut dazu, dass sich in folgenden Generationen wieder die Fähigkeit entwickelte Zitronensäure zu nutzen.

Evolution of increased cell size in all twelve populations
All twelve of the experimental populations show an increase in cell size, and in many of the populations, a more rounded cell shape. 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.

Auch hier zeigen sich also entsprechende Mutationen und Selektionen, wenn diese auch nicht so komplex sind, wie die zuvor aufgetretene Mutation. Dabei ist zu bedenken, dass die hier vorgenommene Selektion natürlich sehr speziell war und daher damit zu rechnen ist, dass die Selektion in eine sehr spezielle Richtung erfolgt. Das ist ähnlich wie bei Zuchtputen, die teilweise soviel Brustfleisch haben, dass sie in der Freiheit nicht lebensfähig wären.

Continued increase in fitness

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.

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.

Es kommt also zu einer fortwährenden Anpassung, eben durch Mutation und Selektion. Das ist insofern auch genau das, was die evolutionäre Theorie voraussagt.