mutations of bacteria from virus sensitivity to virus resistance s. e. luria and m. delbrück

Mutations of Bacteria From Virus Sensitivity to Virus Resistance

S. E. Luria and M. Delbrück

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




• Variance

• Mutation rate

• Conclusions


Bacteria response to bacteriophageWhen 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 survivalDo 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

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


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 survival1. Genetic mutation:The proportion of resistant bacteria increases with timeResistant bacteria will occur as groups of closely related individuals – non-Poisson distribtion


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


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




• 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) – non-Poisson 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…

C C …

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

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):

0t

tN N e

Total number of potential survivors before the attack

m tta N

Mutation hypothesis:

Hereditary acquired immunity:

am – probability density to mutate

a ta N aa – probability density to survive the contact with bacteria

/ m td dt a N Growth rate:at t=0 ρ=0 Total number:

(fixed proportion)

(the proportion grows)

The variance in the mutation hypothesis

2 2var m tCa N

The average number of resistant bacteria in each culture:

ln( )m t t mr a N N CaNt – number of all bacteria at time t, C – number of similar cultures, and 0ln( )t mN Ca t t

The average compared to the variance:

var/ / ln( )t m t mr N Ca N Ca

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

p0 – is the fraction of cultures showing no mutation

0

0

ln( )m

t

pa

N N

N0 and Nt – initial numbers of bacteria and at time t



• Results

• Theoretical model and experiment

• Variance

• Conclusions


• Mutation rate

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: varianceThe number of resistant bacteria in series of similar cultures

In every experiment the fluctuation of thenumbers of resistant bacteria is much higher than could be accounted for by the sampling errors and in conflict with theexpectations from the hypothesis of acquired immunity

Compare variance to the average

Results: mutation rateValues of mutation rate from different experiments

Average mutation rate: 2.45×10-8



• Results• Theoretical model

• Variance

• Comparing experimental and theoretical results

• Mutation rate

• Conclusions

• Proposed mechanisms: short overview of Luria and M. Delbrück’s work

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!

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)

mutations of bacteria from virus sensitivity to virus resistance s. e. luria and m. delbrück

Documents

mutations of bacteria

ability of bacteria

time resistant bacteria

attack slide

mechanisms of survival

proposed mechanisms

virus slide

delbrck slide