Transcript Document

Experimental Molecular Evolution
Evolution of bacterial resistance to antibiotics
D. M. Weinreich, N. F. Delaney, M. A. DePristo & D. L. Hartl.
2006. Darwinian Evolution Can Follow Only Very Few
Mutational Paths to Fitter Proteins. Science 312: 111-114.
Resistance to ß-lactam antibiotics (e.g., penicillin) is
mediated by ß-lactamase, which hydrolyses and
inactivates these drugs.
5 point mutations in ß-lactamase jointly increase
resistance to ß-lactam antibiotics by a factor of
~100,000. These consist of four missense mutations
(A42G, E104K, M182T, G238S) and one 5'
noncoding mutation (g4205a).
5 mutations must occur for the resistant allele TEM*
to evolve from the wild type allele TEMwt.
There are 5! = 120 mutational trajectories to evolve
TEM* from TEMwt.
Experimental Results:
102 of the 120 mutational trajectories from TEMwt to
TEM* are selectively inaccessible.
Most resistance evolved through 10 mutational
trajectories.
Tree of Life
Mesophile = 20-40°C
Thermophile = 45-75°C Hyperthermophile ≥ 80°C
Hypothesis: Last Universal Common Ancestor (LUCA) was
hyperthermophilic (>80 °C), lived in hydrothermal vents (black
smokers)
Reconstructing the past from the present
Proto-Indoeuropean >3000 B.C.
*snig wh-
nix, nivus
  
snaiws snaw
Old
P russian
Church
Slavonic
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snow
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noif
snœr
Old High Middle
German English
Old Fr.
Old Norse
x
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tu
ch sh
ea Iri
sn e h
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ne Fr
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g an
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Slavic
Greek
Gothic Old English
sneo
Old Irish
Latin
P rot o-Germanic
snegu
snechte
Romance
Ce ltic
Reconstruction says something about the Proto-Indoeuropeans
They lived where it snowed.
Elongation Factor-Tu: G-protein involved in translation
Used to elucidate ancient evolutionary relationships
EF-Tu is thermostable in thermophilic organisms, not
in mesophilic organisms
EF-Tu from thermophiles is not optimally functional at
mesophilic temperatures
Linear relationship between optimal binding
temperature of EF protein and optimal growth
temperature of the host organism.
Maximum Likelihood Tree
ML-Stem
Alternative Tree
Outgroup
Outgroup
Thermotogale
Thermus
Thermotogale
Actinobacteria
Actinobacteria
Cyanobacteria
Bacillus
Bacillus
Green Sulfur
Spirochaete
Green Sulfur
Cyanobacteria
Thermus
Spirochaete
Proteobacteria
Proteobacteria
Maximum Likelihood Tree
ML-Stem
Alternative Tree
Outgroup
Outgroup
Thermotogale
Thermus
Alt-Stem
Actinobacteria
Thermotogale
Actinobacteria
Cyanobacteria
Bacillus
Bacillus
Green Sulfur
Spirochaete
Green Sulfur
Cyanobacteria
Thermus
Spirochaete
Proteobacteria
Proteobacteria
Maximum Likelihood Tree
ML-Stem
Alternative Tree
Outgroup
Outgroup
Thermotogale
Thermus
Alt-Stem
Actinobacteria
Thermotogale
Actinobacteria
Cyanobacteria
ML-Meso
Bacillus
Bacillus
Green Sulfur
Spirochaete
Green Sulfur
Cyanobacteria
Thermus
Spirochaete
Proteobacteria
Proteobacteria
Synthesizing Ancestral Proteins
Generate overlapping primer pairs, extended using PCR
(Each primer = 50 bases, with 20 base overlap)
Gene inserted into cloning vector and sequenced
Removed from cloning vector, inserted into expression vector and sequenced
again
Transformed into expression host (E. coli, ER2566), induced with IPTG
This results in the translation of a fusion construct containing:
- Chitin Binding Domain
- Intein
- EFTu gene
EF-Tu Antibody
111 kDa
66 kDa
45 kDa
Precursor
CBD-Intein
EF-Tu
Relative amount of [ 3 H] GDP Incorporation
1
E. coli
0.8
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
Relative amount of [ 3 H] GDP Incorporation
1
ML-meso
E. coli
0.8
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
Relative amount [ 3 H] GDP Incorporation
1
Thermus
0.8
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
100
Relative amount [ 3 H] GDP Incorporation
1
Thermus
ML-stem
0.8
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
100
Relative amount [ 3 H] GDP Incorporation
1
Thermus
Alt-stem
0.8
ML-stem
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
100
Relative amount [ 3 H] GDP Incorporation
Thermus
Alt-stem
ML-stem
Thermotoga
1
0.8
0.6
0.4
0.2
0
20
30
40
50
60
o
C
70
80
90
100
Hydrothermal
Vents:
Broad Range of
Temperatures Across
Narrow Area
Thermal
Hot Springs:
Narrow Range of
Temperatures Across
Broad Area
Consistent with
ancient EFs
~65ºC
Molecular
Breeding
selection
+
breeding
Very variable
population
Monomorphic
population
selection
+
breeding
Less variable
population
No
population
How to create novel
variation
1. Mutation
a. Random
b. Directed
2. Recombination
Mutations
occur at low
frequencies
and are
mostly
deleterious.
Directed
mutations are
useful if we
know a priori
which
sequence will
accomplish
which task.
Recombination
produces a lot of
functional
variation.
Willem P. Stemmer
The Protocol…
1. Identify a product that can be
improved.
… and sold with no
controversy.
Laundry detergents contain the following
active enzymes:
Protease — removal of protein stains
Amylase — removal of starchy stains
Lipase — removal of greasy stains
Peroxidase — bleaching
Cellulase — softening
2. Select a gene that may improve the
product.
3. Obtain homologous genes from
diverse sources.
4. Mix “parental” genes in a solution.
5. Fragment the genes in a number of
different ways.
6. Heat the solution so the fragments become
single stranded.
7. Cool the solution so that the gene fragments
reanneal at sites of complementarity, thus, creating
novel recombinations.
8. The novel recombinations are extended, so
that double-stranded heteroduplex DNA
molecules are created.
8. The recombination process is repeated…
9. … until full-length double-stranded
heteroduplex DNA molecules are created.
10. The result is a library of novel full-length
genes which have different combinations of
characteristics from the “parental” genes.
etc…
11. Test each recombinant for the desired property.