MUTATIONS OF BACTERIA FROM VIRUS SENSITIVITY TO VIRUS RESISTANCE
S.E LURIA and M. DELBRUCK
Mutation refers to any heritable change in the DNA sequence. The errors and damages that repaired by DNA are not mutations. This genetic changes needs to be permanent and should transfer to the second generation.
There are three main types of mutants;
- Auxotrophic mutants; a mutant strain of microorganism that will proliferate only when the medium is supplemented with some specific substance not required by wild-type organisms.
- Conditional lethal mutants; these mutants can cause lethality under one condition (the restrictive or non-permissive condition) but not another (the permissive condition).
- Temperature sensitive mutants; mutants which are able to grow at certain low temperatures(32°C), which is referred to as the permissive temperature, but are unable to grow at higher temperatures (39°C), which is referred to as the nonpermissive temperature.
- Cold-sensitive mutants; mutant cells with proteins that fail to function at lower temperatures
- Nonsense mutations; mutations that changed the three nonsense codons (UGA, UAG or UAA)
- Resistant mutants; mutants that develop resistance mechanisms against to a substance (drugs, phages, antimicrobials) that kills or inhibits the growth of a bacterium.
Mutation rate; it is a measure of the rate at which various types of mutations occur over time.
Luria and Delbruck Experiment
Back at that time, many microbiologists believed that bacteria were carrying genetic heritance and having mutations as a result of the adaption to the new environment whereas some others believed these mutations were accruing randomly. However in the year of 1943, Luria and Delbruck wondered about the nature of mutations. Are mutations spontaneous? Or do they occur in response to environmental conditions?
In the early 1990s, it was a well-known fact that bacterial viruses had antimicrobial effects. Some of the bacterial colonies that survive from this attack were called “resistant” whereas the succumbed ones called “sensitive”. The mechanism beyond this selection was unknown, however In the 1920s there was two hypothesis suggested regarding this issue;
Hypothesis one was the direct action of the virus reduces induced the resistant variants. The second hypothesis was; the resistant bacterial variants are produced by mutation in the culture before the virus was added. However, none of them was proved at that time. Basically, in terms of the experiment, a bacterial culture from a single cell plated with the virus in access and upon incubation, a small fraction of would survive. So, the attack of the virus prevailed the development of small numbers of colonies. By discovering these little survival resistant colonies;
Luria and Delbrück suggested two main hypothesis; this resistance was occurring due to mutations which are independent of a virus or there was an acquired immunity (hereditary predisposal individuals or after the infection they gained immunity) in the resistant cells. The first hypothesis had nothing to do with the virus since the mutation supposed to happen sometimes randomly there, every offspring of mutants supposed to be resistant unless a reverse mutation occurs. The second hypothesis which had a smaller chance; had something to do with the virus since it was the reason for the resistance. So for both hypothesis each offspring of a tested bacterium would survive after the virus exposure. However they would be distinguished by two main differences;
- A single colony would be expected on the mutation hypothesis while a random distribution of resistant would be expected on the acquired hereditary immunity hypothesis.
- If the resistance was due to mutation, the proportion of the resistant bacteria supposed to increase by time.
They observed some fluctuations (thereby their experiment also named the fluctuation test experiment) about the number of resistant cells after a couple of basic experiments and they said the controlled conditions were the same for all experiments. They couldn’t understand these fluctuations at the beginning. And they assumed that the large fluctuations might be the result of the mutation hypothesis.
The clues that they achieved about the growth of the resistance cells; they wanted to predict the number of the mutant cells will be achieved by time. To have such probability analyzes they needed to use a binomial distribution because the result of this virus exposure could be only; survive or die. However, since the survival chance is not equal, as a matter of fact it is much lesser than the death chance therefore, they used poisson distribution instead of the binomial. At the end, they achieve an equation for the mutation rate by grouping mutations according to the bacterial generations.
They used Escherichia coli B cells and bacterial virus α, they used nutrient broth and agar plates and asparagine-glucose synthetic medium. The division time for the medium was 35 min whereas for the broth it as 19 min. In the synthetic medium that they used the pH had increased from 7 to 5 by the incubation time. They also used only nutrient agar plates for plating the bacterial cells and exposing them to the virus. Since the lysis was very fast on the plate due to the virus, it was very easy to detect the resistant colonies. They also run a test to see how reliable the plating method by parallel plating is. The results were as expected. They also test the number of resistant bacteria in different samples from the same cultures. The hypothesis was; if the resistance was due to the mutation the variance supposed to be much greater from the average whereas if it is due to immunity the variance supposed to close to the mean. They run 100 sample by using aerated broth, regular broth, and synthetic medium and compared the results with their expected numbers. They inoculated a small number of bacteria (Escherichia coli) into separate culture tubes. After a period of growth, they plated equal volumes of these separate cultures onto agar containing the virus. If resistance to the virus in bacteria were caused by an induced activation in bacteria if resistance were not due to hereditary immunity, then each plate supposed to contain roughly the same number of resistant colonies. Assuming a constant rate of mutation, they hypothesized that if mutations occurred after and in response to exposure to the selective agent, the number of survivors would be distributed according to a Poisson distribution with the mean equal to the variance. However what they observed was; instead, the number of resistant colonies on each plate varied drastically: the variance was considerably greater than the mean. This could be caused by many simple reasons that would cause such results e.g. t0, physiological state of the bacteria or sudden transition from sensitivity to resistance. However of these were the case, they wouldn’t expect to see a very high portion of cultures to be different, in the fig. 2 the number of jackpots* (>9 resistant bacteria) far exceeded that expected by chance.
Luria and Delbruck’s experiments showed unequivocally that mutations were spontaneous. By these experiments, they proved that the resistance occurred due to mutations independently of the action of the virus and with the math they did, they found how to calculate/ estimate the mutation rate.
*They called this difference “jackpot” cause they say” The situation is similar to the operation of a (fair) slot machine, where the average return on a limited number of plays is probably considerably less than the input, and improbably, when the jackpot is hit, the return is much bigger than the input.”