Luria Delbrück experiment

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The Luria-Delbrück experiment (also known as fluctuation test ) is an experiment devised and carried out by Salvador Edward Luria and Max Delbrück , which was published in 1943 and which was part of the work for which Luria and Delbrück together with Alfred Hershey received the Nobel Prize in 1969 Physiology or Medicine .

The experiment, in which the resistance of bacteria to bacteriophages was investigated, showed that random mutations in bacteria occur spontaneously and thus also in the absence of selection mechanisms , and refuted the alternative hypothesis that these occur in response to changed conditions in the environment. With the help of a mathematical model developed by Delbrück, the hypotheses could be tested in a quantitative way.

In addition, Luria and Delbrück were able to determine the mutation rate of the bacteria with the help of some model assumptions . For this purpose, fluctuation tests are still carried out today.

initial situation

At the time of the experiment, it had long been known that a bacterial culture that is exposed to a deadly bacteriophage is initially severely decimated by it. After a while, the apparently eliminated culture grows again because a resistant variant of the bacterium has formed that can multiply unhindered. However, the mechanism of mutations was unknown in the early 1940s, especially whether they occur before the virus was added or in response to it. Little was known about the molecular basis of inheritance either ; DNA as the carrier of genetic information had not yet been discovered.

At that time Luria was conducting experiments on bacteriophages. One of his problems here was that the number of bacteria that had become resistant to phages was subject to very large fluctuations. Luria got the idea for the experiment while looking at a colleague at a slot machine . The machine usually kept the player's stake and in rare cases paid out the entire jackpot. Luria realized that the fluctuations should not be seen as an obstacle to be overcome, but should be made the object of analysis themselves. The trained physicist Max Delbrück, to whom Luria reported his idea, then worked out the theoretical basis of the experiment.

Experiment description

Two possible experimental results: (A) If the mutations are induced by the addition of the phage, a similar number of resistant bacteria will appear in each culture. (B) If mutations occur spontaneously and therefore independent of the presence of phages, strong fluctuations are to be expected. In the experiment it was shown that type (B) distributions occur in nature.

A large number of separate cultures of the bacterium Escherichia coli were produced in the experiment . Initially, 50 to 100 non-resistant bacteria multiplied in a nutrient solution until colonies of around 10 9 bacteria developed. Each of the cultures was then exposed to a high concentration of bacteriophage T1, which is lethal to non-resistant bacteria, on nutrient plates. After a waiting period of 24 and 48 hours, the number of colonies of (resistant) bacteria was determined and the resulting distribution was determined from the data from many samples .

The main aim of the experiment was to distinguish between two alternative hypotheses relating to the mutation: According to the first hypothesis, the development of resistance in bacteria occurs in response to the attack by the phages. With a low probability, individual bacteria can develop a resistance to the phages independently of one another, which they pass on to their offspring. In this case the expected distribution of the cultures corresponds to a Poisson distribution . Their fluctuations are relatively small, since the variance of a Poisson distribution is equal to its mean. If this “acquired immunity” hypothesis were correct, the results would look like the left column of the diagram.

According to the second hypothesis, there is a low probability that resistance-building mutations occur continuously, i.e. even before the bacterial culture has been attacked by the phages. In most cultures, these mutations appear late, when the initial 50 to 100 bacteria have already multiplied strongly. The resistant bacteria then have little time to multiply, and only a few resistant bacteria are present at the time of the bacteriophage attack, which leads to low measured concentrations. In a few cultures, however, resistance-building mutations occur early on, so that after prolonged exponential growth, a large number of bacteria survive the attack by the phages (see right-hand column of the diagram). As a result, the measured distribution has a variance that is many times greater than its mean value. There are rare events ("jackpot") where extremely large numbers of bacteria are measured. A mathematical model developed by Delbrück made it possible to specify a formula for the variance of the expected distribution and to determine the mutation rate approximately.

The experiment clearly confirmed the correctness of the second hypothesis. The measured variance of the distribution was many times greater than the mean value and agreed well with the model predictions. This showed the existence of permanent random mutations in bacteria.

Aftermath

The fluctuation test has often been interpreted as showing the validity of Darwin's theory of evolution in bacteria and refuting Lamarckian theories. Luria herself had described bacteriology as the "last bulwark of Lamarckism".

Luria and Delbrück themselves were initially cautious about the generalizability of their results. Their results were confirmed in the following years by further experiments, in which, for example, instead of resistance to bacteriophages, resistance to penicillin or X-rays was tested.

While Luria and Delbrück only calculated the mean value and variance of the distribution to be expected according to the theory of spontaneous mutations from their model, Lea and Coulson succeeded in 1949 in deriving a mathematical expression for the distribution itself. It is called the Luria-Delbrück distribution and is still used today to determine mutation rates.

In the late 1980s, the generality of the randomness of mutations was challenged by work by John Cairns and co-workers. In contrast to Luria and Delbrück, Cairns investigated cases of non-lethal selection (i.e. the bacteria can survive under selection pressure but cannot multiply) and found that mutations favorable to the bacteria occurred significantly more frequently than could be explained by random mutations. After a lengthy, controversial scientific debate about this phenomenon, known as adaptive mutation , it is now known that various mechanisms exist by which organisms can increase the frequency of mutations in response to external selection pressure, for example through the formation of error-prone polymerases . However, the existence of targeted mutations in response to external selection pressure could not be shown.

See also

literature

  • Luria, SE, Delbrück, M .: Mutations of bacteria from virus sensitivity to virus resistance. In: Genetics. Volume 28, Number 6, November 1943, pp. 491-511, PMID 17247100 , PMC 1209226 (free full text).

Individual evidence

  1. ↑ EP Fischer: Max Delbrück. Genetics 177, 673-676 (2007)
  2. ↑ "Last stronghold of Lamarckism" G. Stent: The 1969 Nobel Prize for Physiology or Medicine . Science 24, 166 (1969)
  3. ↑ Luria, Delbrück, p. 509
  4. ↑ DE Lea, CA Coulson: The distribution of the number of mutants in bacterial populations J. Genet 49, 264–285 (1949)
  5. ↑ WA Rosche, PL Foster: Determining Mutation Rates in Bacterial Populations. Methods 20, 1 (2000)
  6. ↑ J. Cairns, J. Overbaugh S. Miller: The Origin of mutants , Nature 335, 142-145 (1988)
  7. ↑ SM Rosenberg: Evolving responsively: adaptive mutation , Nature Reviews Genetics 2, 504-515 (2001)

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