1. Mutations of Bacteria From Virus Sensitivity to Virus Resistance S. E. Luria and M. Delbrück

2. Outline Introduction Bacteria response to bacteriophage Results Theoretical model and experiment Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work Variance Mutation rate Conclusions

3. Outline Introduction Bacteria response to bacteriophage Results Theoretical model and experiment Variance Mutation rate Conclusions Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work

4. Bacteria response to bacteriophage When bacteria are mixed with bacteriophage:

5. Bacteria response to bacteriophage When bacteria are mixed with bacteriophage:

6. Bacteria response to bacteriophage When bacteria are mixed with bacteriophage:

7. Bacteria response to bacteriophage If about a billion bacteria mixed with a particular toxin, nearly all of the bacteria are killed. A few will survive and give rise to colonies that are permanently and specifically resistant to that particular toxin

8. Proposed mechanisms for survival Do the bacteria have genes and how do they survive an attack? Mendelian mechanism: hypothesis of mutation Lamarckian mechanism: hypothesis of acquired hereditary immunity Small probability of developing resistance upon contact with phage, no genetic component

9. Proposed mechanisms for survival If resistance is produced by physiological adaptation: 1. The proportion of resistant bacteria will stay constant during the attack 2. Resistant bacteria occur as separate and scattered individuals (every resistance is an independent event with no genetic component) Not the case: the proportion of the resistants grows during the attack

10. Proposed mechanisms for survival The researchers were puzzled by ability of bacteria to respond rapidly and adaptively to changes in the environment In 1943, Salvador E. Luria and Max Delbrück showed that apparent examples of Lamarckian inheritance were actually due to true genetic mutation in 1946 Edward Tatum and Joshua Lederberg showed that both linkage and recombination could be detected in bacteria

11. Proposed mechanisms for survival 1. Genetic mutation: The proportion of resistant bacteria increases with time Resistant bacteria will occur as groups of closely related individuals – non-Poisson distribtion

12. Proposed mechanisms for survival 2. Acquired hereditary immunity: Resistant bacteria occur as separate and scattered individuals (every resistance is an independent event) Poisson distribution of resistant bacteria Immunity only upon the interaction with the virus

13. Proposed mechanisms for survival Two experimental methods are available: 1. See if the proportion of resistants increases over time 2. Examine groups of related bacteria (colonies) to see if the resistance is correlated with genetic descent

14. Proposed mechanisms for survival Adaptation hypothesis: each resistant occurs as a separate, random event. No clones of resistants before the attack. Poisson distribution of survivors Mutation – grows of clones of resistants before the attack. Non-Poisson results

15. Outline Introduction Bacteria response to bacteriophage Results Theoretical model and experiment Variance Mutation rate Conclusions Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work

16. Hypothesis of mutation The bacteria had the resistance ahead of time of the attack. No interaction with virus. No new mutant trees (colonies) during the attack Acquired hereditary immunity Bacteria gets immune during the attack. Mutant trees (colonies) appear only during the attack

17. The main difference between the theories Mutation hypothesis: correlation between the mutants (few colonies before the attack) – non- Poisson distribution Acquired hereditary immunity: random distribution of resistants (many colonies formed during the attack) – Poisson distribution Look at variances

18. Experiment Start from one bacterium. Grow it for a few generations Put the same amount in a number of Petri-dish filled with virus Count how many bacteria survived (count colonies)

19. Experiment Grow bacteria to a few generations in different flasks Spread equal amount from each flask into dishes with the virus D1D2D3D4D5Dn … CC … After 24-48 hours count colonies found in the dishes:

20. Total number of bacteria The number N t of bacteria in a growing culture follows the equation (time unit: the average division time of the bacteria/ln 2):

21. Total number of potential survivors before the attack Mutation hypothesis: Hereditary acquired immunity: a m – probability density to mutate a a – probability density to survive the contact with bacteria Growth rate: at t=0 ρ=0 Total number: (fixed proportion) (the proportion grows)

22. The variance in the mutation hypothesis The average number of resistant bacteria in each culture: N t – number of all bacteria at time t, C – number of similar cultures, and

23. The average compared to the variance: The ratio between variance and average >> 1, if N t Ca m >> 1 This will be measured in experiment. It must give var/r >> 1 for non-Poisson distribution

24. Mutation rate p 0 – is the fraction of cultures showing no mutation N 0 and N t – initial numbers of bacteria and at time t

25. What is inoculum, explain the experiment

26. Outline Introduction Bacteria response to bacteriophage Results Theoretical model and experiment Variance Conclusions Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work Mutation rate

27. Results The two hypotheses lead to radically different distributions of the number of the resistant bacteria in a series of similar cultures: Hypothesis of acquired immunity: variance equal to the average The mutation hypothesis: variance much greater than the average

28. Results: variance The number of resistant bacteria in series of similar cultures In every experiment the fluctuation of the numbers of resistant bacteria is much higher than could be accounted for by the sampling errors and in conflict with the expectations from the hypothesis of acquired immunity Compare variance to the average

29. Results: mutation rate Values of mutation rate from different experiments Average mutation rate: 2.45×10 -8

30. Outline Introduction Bacteria response to bacteriophage Results Theoretical model Variance Comparing experimental and theoretical results Mutation rate Conclusions Proposed mechanisms: short overview of Luria and M. Delbrück’s work

31. Conclusions The resistance is due to mutation, independent of virus The average mutation rate is 2.45×10 -8 ; as rare as in higher organisms Random gene mutation followed by selection is responsible for the adaptation of bacteria to virus

32. Toda raba!

33. Artificial Nano “T4 Bacteriophage” Size of the artificial nano “T4 Bacteriophage” 10× of the real virus Made of Diamond-like Carbon by Reo Kometani & Shinji Matsui (University of Hyogo) by FIB-CVD (focused ion beam - chemical vapor deposition)