Learning new tricks

Evolution

As part of a study, scientists replayed the course of evolution by observing bacteria rapidly multiply through 55,000 generations in just 25 years. Over 55,000 generations, these e. coli evolved generally, but at the 31,000th generation, one of the 12 lines of e. coli began to use citrate as nutrient, as if it were in oxygen-free conditions, writes S Ananthanarayanan

Darwin’s theory of evolution of species provided the device of natural selection, by which specific random variations in generations were favoured for survival. Gregor Mendel’s work was on the mechanics of inheritance and he showed that traits were passed on in units, called genes. There have been discussions on whether the great specialisation that has been seen in evolution, like development of flight or of organs like the eye, could come about through random processes. Should there not be some ‘direction’ to the variations that were selected?

Darwin himself had said, “Natural selection acts only by taking advantage of slight successive variations; she can never take a great and sudden leap, but must advance by short and sure, though slow steps...If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down.” But evolution takes so long in happening, with evidence only in prehistoric fossil records, that there is no way to verify what the intermediate steps actually were. Zachary D Blount, Jeffrey E Barrick, Carla J Davidson and Richard E Lenski of Michigan, Texas and Calgary, Canada report in the journal, Nature, that they replayed the course of evolution in quick time, by observing bacteria rapidly multiply through 55,000 generations in just 25 years.

Escherichia coli

The bacterium, e. coli is an organism, just two micros in size, found in the intestines of warm blooded animals. The species came into existence about 100 million years ago, along with warm blooded animals, and has remained unchanged since over 20 million years.

The organisms were discovered by Theodor Escherich, a German paediatrician, in 1885, who called them bacterium coli, as they were found in the colon. E. coli, as a simple and easily grown laboratory bacterium, has been a very useful base of research in biotechnology and microbiology. Its special importance is that it is a fully equipped living organism and, more important, that it reproduces every few minutes. This pace of reproduction is to create 75 generations in a day and over 2,000 generations in a year.

Richard E Lenski, co-author of the paper, had started this long running project in experimental evolution in 1988. A group of fast-growing e. coli, descended from the same parent cell, was used to found 12 independent populations in 12 different culturing flasks, containing the same simple culture medium, which had the components essential for growth and glucose as the sole source of carbon. Every day, for nearly 25 years, a sample from each flask has been transferred to a fresh flask, to start a new culture. What we have now is thus 12 lines of some 9,000 flasks with about 55,000 generations of e. coli.

In addition to fresh cultures, every few days, samples from the flasks were also stored at −80°C. We thus have with us the time frozen samples of e coli as they had evolved at various times along these 25 years. Unlike fossil records, however, these samples of past times can be thawed and revived, for study and further generation.

Over these 55,000 generations, the samples of e. coli did evolve for greater fitness, generally. But at about 31,000 generations, a remarkable development was noticed. The growth medium in which the e. coli were cultured, where glucose was the source of carbon, contained citrate as the agent that enabled uptake of iron, which is usually insoluble, by e. coli. While citrate also contains carbon, which e. coli can use in oxygen-free conditions, citrate is not a source of carbon in the well aerated conditions of the experiment. But what was seen, at the 31,000th generation, was that one of the 12 lines of e. coli began to use citrate as nutrient, as if it were in oxygen-free conditions!
This development was different from the usual, single step evolution of fitness that was seen generally.

To gain the ability to use citrate in the presence of oxygen (a variety of e. coli called cit+) is on par with an evolutionary leap, on the lines of, although not equal to developing flight, for instance. The experiment had thus thrown up an instance of the very kind of evolutionary change whose mechanism was under question. But the design of the experiment allowed investigation, up the time-line, into the earlier stages, to see where exactly the crucial change took place. One thing was clear, that the ability to use citrate was new trait altogether – it was a modification of an existing trait – with refinement of the conditions in which it could be expressed. But where and what changes had taken place were to be identified.

Action replay

The researchers went back in time with the help of the frozen samples and resuscitated ancestors of the current cit+ population. The resuscitated ancestors were then cultured, to see which ones had the ability to evolve into cit+. The cut-off would then identify the single change which provided the ability for this evolution. Investigation showed that the cit+ evolution potential existed only in a few, most recent non-cit+ samples. What was the change at these stages, which were the ‘potentialised’ stages, was difficult to trace, but when cit+ did appear, ie, the actualisation step, was easier to pin down.

The action of citrate entering the cell, which is what cit+ cells permitted, takes place because of a protein which provides the molecular handle for this action. The researchers therefore analysed the different e. coli DNA at the region which carries the gene citT, which enables generation of that protein. It was found that in the original e. coli cells, citT was located downstream of citG, another gene related to citrate use, and also to rnk, an unrelated gene that affects energy metabolism. But in cit+ cells, the genes were re-arranged, with rnk and citG being fused, and rnk being effective in allowing the expression of citT and citG in the presence of oxygen.

In fact, the study has shown that a single copy of the genetic rearrangement was not sufficient to generate cit+, but an array of two to nine copies were needed. It was cells that inherited the fusion of rnk and citG and then followed through with amplifying mutations that grew to be cit+.

The discovery, as a result of well designed, workmanlike investigation into how evolution works is confirmation that major innovations can be explained by the working of gradual, micro-evolutionary stages of potentialisation, followed by actualisation and amplification.

The work amounts to providing a mechanism of how apparently discontinuous steps in evolution come about through the operation of a series of single step genetic changes, an answer to anti-evolutionists.

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