Transcript Mutations of Bacteria From Virus Sensitivity to Virus

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
• Mutation rate
• Conclusions
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
When bacteria are mixed with bacteriophage:
Bacteria response to bacteriophage
When bacteria are mixed with bacteriophage:
Bacteria response to bacteriophage
When bacteria are mixed with bacteriophage:
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
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 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 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 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 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 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 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
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
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
Mutation hypothesis: correlation between the
mutants (few colonies before the attack) – nonPoisson distribution
Acquired hereditary immunity: random
distribution of resistants (many colonies formed
during the attack) – Poisson distribution
Look at variances
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)
Experiment
Grow bacteria to a few generations
in different flasks
Spread equal amount from each flask into dishes with the virus
D1
D2
D3
D4
D5 … Dn
After 24-48 hours count colonies found in the dishes:
C
C
…
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):
Nt  N0e
t
Total number of potential survivors before the
attack
Mutation hypothesis:
d  / dt  am Nt  
at t=0 ρ=0
Total number:   ta N
m t
Growth rate:
(the proportion grows)
am – probability density to mutate
Hereditary acquired immunity:
  aa Nt
(fixed proportion)
aa – probability density to survive the contact with bacteria
The average number of resistant bacteria in
each culture:
r  am Nt ln( Nt Cam )
Nt – number of all bacteria at time t, C – number of
similar cultures, and ln( Nt Cam )  t  t0
The variance in the mutation hypothesis
var  Cam N
2
2
t
The average compared to the variance:
var/ r  Nt Cam / ln( Nt Cam )
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
ln( p0 )
am  
Nt  N 0
p0 – is the fraction of cultures showing no mutation
N0 and Nt – initial numbers of bacteria and at time t
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
• 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
The number of resistant bacteria in series of similar cultures
variance
InCompare
every experiment
theto
fluctuation
of the
the average
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
Values of mutation rate from different experiments
Average mutation rate: 2.45×10-8
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
• 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
 Toda raba! 
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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)