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Mutations of Bacteria From Virus Sensitivity to Virus Resistance. S. E. Luria and M. Delbrück. Outline. Introduction. Bacteria response to bacteriophage. Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work. Theoretical model and experiment. Results. Variance.

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mutations of bacteria from virus sensitivity to virus resistance

Mutations of Bacteria From Virus Sensitivity to Virus Resistance

S. E. Luria and M. Delbrück

outline
Outline
  • Introduction
  • Bacteria response to bacteriophage
  • Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work
  • Theoretical model and experiment
  • Results
  • Variance
  • Mutation rate
  • Conclusions
outline3
Outline
  • Introduction
  • Bacteria response to bacteriophage
  • Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work
  • Theoretical model and experiment
  • Results
  • Variance
  • Mutation rate
  • Conclusions
bacteria response to bacteriophage
Bacteria response to bacteriophage

When bacteria are mixed with bacteriophage:

bacteria response to bacteriophage5
Bacteria response to bacteriophage

When bacteria are mixed with bacteriophage:

bacteria response to bacteriophage6
Bacteria response to bacteriophage

When bacteria are mixed with bacteriophage:

bacteria response to bacteriophage7
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
proposed mechanisms for survival
Proposed mechanisms for survival

Do the bacteria have genes and how do they survive an attack?

  • Small probability of developing resistance upon contact with phage, no genetic component
  • Lamarckian mechanism: hypothesis of acquired hereditary immunity
  • Mendelian mechanism: hypothesis of mutation
proposed mechanisms for survival9
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

proposed mechanisms for survival10
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
proposed mechanisms for survival11
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

proposed mechanisms for survival12
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

proposed mechanisms for survival13
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

proposed mechanisms for survival14
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
outline15
Outline
  • Introduction
  • Bacteria response to bacteriophage
  • Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work
  • Theoretical model and experiment
  • Results
  • Variance
  • Mutation rate
  • Conclusions
hypothesis of mutation
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

the main difference between the theories
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

experiment
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)
experiment19

Grow bacteria to a few generations in different flasks

Spread equal amount from each flask into dishes with the virus

D1

C

D2

D3

D4

C

D5

Dn

After 24-48 hours count colonies found in the dishes:

Experiment
total number of bacteria
Total number of bacteria
  • The number Nt of bacteria in a growing culture follows the equation (time unit: the average division time of the bacteria/ln2):
total number of potential survivors before the attack
Total number of potential survivors before the attack

Mutation hypothesis:

Growth rate:

at t=0 ρ=0 Total number:

(the proportion grows)

am – probability density to mutate

Hereditary acquired immunity:

(fixed proportion)

aa – probability density to survive the contact with bacteria

the variance in the mutation hypothesis

Nt – number of all bacteria at time t, C – number of similar cultures, and

The variance in the mutation hypothesis

The average number of resistant bacteria in each culture:

slide23
The average compared to the variance:

The ratio between variance and average >> 1, if NtCam >> 1

This will be measured in experiment. It must give var/r >> 1 for non-Poisson distribution

mutation rate
Mutation rate

p0 – is the fraction of cultures showing no mutation

N0and Nt – initial numbers of bacteria and at time t

outline25
Outline
  • Introduction
  • Bacteria response to bacteriophage
  • Proposed mechanisms of survival: short overview of Luria and M. Delbrück’s work
  • Theoretical model and experiment
  • Results
  • Variance
  • Mutation rate
  • Conclusions
results
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

results variance
Results: variance

The number of resistant bacteria in series of similar cultures

Compare variance to the average

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

results mutation rate
Results: mutation rate

Values of mutation rate from different experiments

Average mutation rate: 2.45×10-8

outline29
Outline
  • Introduction
  • Bacteria response to bacteriophage
  • Proposed mechanisms: short overview of Luria and M. Delbrück’s work
  • Theoretical model
  • Results
  • Variance
  • Comparing experimental and theoretical results
  • Mutation rate
  • Conclusions
conclusions
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
slide32

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)