Mutations of Bacteria From Virus Sensitivity to Virus Resistance

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Mutations of Bacteria From Virus Sensitivity to
Virus Resistance

• S. E. Luria and M. Delbrück

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Outline

• Introduction

• Bacteria response to bacteriophage

• Proposed mechanisms of survival short overview
  of Luria and M. Delbrücks work

• Theoretical model and experiment

• Results

• Variance

• Mutation rate

• Conclusions

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Outline

• Introduction

• Bacteria response to bacteriophage

• Proposed mechanisms of survival short overview
  of Luria and M. Delbrücks work

• Theoretical model and experiment

• Results

• Variance

• Mutation rate

• Conclusions

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Bacteria response to bacteriophage

• When bacteria are mixed with bacteriophage

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Bacteria response to bacteriophage

• When bacteria are mixed with bacteriophage

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Bacteria response to bacteriophage

• When bacteria are mixed with bacteriophage

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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

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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

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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
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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

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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
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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
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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
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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

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Outline

• Introduction

• Bacteria response to bacteriophage

• Proposed mechanisms of survival short overview
  of Luria and M. Delbrücks work

• Theoretical model and experiment

• Results

• Variance

• Mutation rate

• Conclusions

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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
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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
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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)

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Experiment
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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/ln 2)

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Total number of potential survivors before the
attack
Mutation hypothesis
Growth rate
at t0 ?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
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The variance in the mutation hypothesis
The average number of resistant bacteria in each
culture
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• The average compared to the variance

The ratio between variance and average gtgt 1, if
NtCam gtgt 1
This will be measured in experiment. It must give
var/r gtgt 1 for non-Poisson distribution
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Mutation rate

• p0 is the fraction of cultures showing no
  mutation

N0 and Nt initial numbers of bacteria and at
time t
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• What is inoculum, explain the experiment

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Outline

• Introduction

• Bacteria response to bacteriophage

• Proposed mechanisms of survival short overview
  of Luria and M. Delbrücks work

• Theoretical model and experiment

• Results

• Variance

• Mutation rate

• Conclusions

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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
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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
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Results mutation rate
Values of mutation rate from different experiments
Average mutation rate 2.4510-8
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Outline

• Introduction

• Bacteria response to bacteriophage

• Proposed mechanisms short overview of Luria and
  M. Delbrücks work

• Theoretical model

• Results

• Variance

• Comparing experimental and theoretical results

• Mutation rate

• Conclusions

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Conclusions

• The resistance is due to mutation, independent of
  virus

• The average mutation rate is 2.4510-8 as rare
  as in higher organisms

• Random gene mutation followed by selection is
  responsible for the adaptation of bacteria to
  virus

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? Toda raba! ?
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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)