Experimental Molecular EvolutionExperimental 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.

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.

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.

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

Hypothesis: Last Universal Common Ancestor (LUCA) was hyperthermophilic (>80 °C), lived in hydrothermal vents (black smokers)

Mesophile = 20-40°C Thermophile = 45-75°C Hyperthermophile ≥ 80°C

xx x x x xxx x x x xx

>3000 B.C.

Proto-Germanic

MiddleEnglish

Old English

Old HighGerman

Gothicsnaiws

snow

snaw

sneoChurchSlavonic

snegu

Old Irish

Proto-Indoeuropean

Old Norse

Greekφιν

.Old Fr

Latin

OldPrussian

snechte

œsn rnoif

, nix nivus

α

*snigw -h

xx

Slavic Germanic Romance Celtic

Reconstructing the past from the present

Reconstruction says something about the Proto-Indoeuropeans

They lived where it snowed.

Elongation Factor-Tu: G-protein involved in translationElongation 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.

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.

Proteobacteria

Cyanobacteria

Spirochaete

Green Sulfur

Bacillus

ActinobacteriaThermus

Thermotogale

Outgroup Outgroup

Thermotogale

Thermus

Bacillus

Green Sulfur

Spirochaete

Cyanobacteria

Proteobacteria

Actinobacteria

Maximum Likelihood Tree Alternative Tree

ML-Stem

Proteobacteria

Cyanobacteria

Spirochaete

Green Sulfur

Bacillus

ActinobacteriaThermus

Thermotogale

Outgroup Outgroup

Thermotogale

Thermus

Bacillus

Green Sulfur

Spirochaete

Cyanobacteria

Proteobacteria

Actinobacteria

Maximum Likelihood Tree Alternative Tree

ML-Stem Alt-Stem

Proteobacteria

Cyanobacteria

Spirochaete

Green Sulfur

Bacillus

ActinobacteriaThermus

Thermotogale

Outgroup Outgroup

Thermotogale

Thermus

Bacillus

Green Sulfur

Spirochaete

Cyanobacteria

Proteobacteria

Actinobacteria

Maximum Likelihood Tree Alternative Tree

ML-Stem

ML-Meso

Alt-Stem

Synthesizing Ancestral ProteinsSynthesizing Ancestral Proteins

Generate overlapping primer pairs, extended using PCR (Each primer = 50 bases, with 20 base overlap)

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

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

Precursor

CBD-InteinEF-Tu

111 kDa

66 kDa45 kDa

EF-Tu Antibody

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90

oC

Relative amount of [

3H] GDP Incorporation

E. coli

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90

oC

Relative amount of [

3H] GDP Incorporation

ML-meso

E. coli

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90 100

Thermus

oC

Relative amount [

3H] GDP Incorporation

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90 100

Thermus

ML-stem

oC

Relative amount [

3H] GDP Incorporation

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90 100

Thermus

Alt-stem

ML-stem

oC

Relative amount [

3H] GDP Incorporation

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60 70 80 90 100

ThermusAlt-stemML-stemThermotoga

oC

Relative amount [

3H] GDP Incorporation

HydrothermalVents:

Broad Range of Temperatures

Across Narrow Area

ThermalHot Springs:

Narrow Range of Temperatures

Across Broad Area

Consistent withancient EFs

~65ºC

Molecular Molecular BreedingBreeding

Very variableVery variablepopulationpopulation

MonomorphicMonomorphicpopulationpopulation

selectionselection

++breedingbreeding

Less variableLess variablepopulationpopulation

NoNopopulationpopulation

selectionselection

++breedingbreeding

How to create novel

variation1. Mutationa. Randomb. 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. StemmerWillem P. Stemmer

The Protocol… The Protocol…

1. Identify a product 1. Identify a product that can be improved.that can be improved.

… … and sold and sold with no with no controversy.controversy.

Laundry detergents contain Laundry detergents contain the following active enzymes: the following active enzymes:

Protease — removal of Protease — removal of protein stainsprotein stains

Amylase — removal of Amylase — removal of starchy stainsstarchy stains

Lipase — removal of greasy Lipase — removal of greasy stainsstains

Peroxidase — bleachingPeroxidase — bleachingCellulase — softeningCellulase — softening

Laundry detergents contain Laundry detergents contain the following active enzymes: the following active enzymes:

Protease — removal of Protease — removal of protein stainsprotein stains

Amylase — removal of Amylase — removal of starchy stainsstarchy stains

Lipase — removal of greasy Lipase — removal of greasy stainsstains

Peroxidase — bleachingPeroxidase — bleachingCellulase — softeningCellulase — softening

2. Select a gene that may 2. Select a gene that may improve the product.improve the product.

3. Obtain homologous 3. Obtain homologous genes from diverse genes from diverse sources.sources.

4. Mix “parental” genes in a solution.4. Mix “parental” genes in a solution.

5. Fragment the genes in a 5. Fragment the genes in a number of different ways.number of different ways.

6. Heat the solution so the 6. Heat the solution so the fragmentsfragments become single stranded. become single stranded.

7. Cool the solution so that the 7. Cool the solution so that the gene fragments reanneal at sites of gene fragments reanneal at sites of complementarity, thus, creating complementarity, thus, creating novel recombinations. novel recombinations.

8. The novel recombinations are 8. The novel recombinations are extended, so that double-extended, so that double-stranded heteroduplex DNA stranded heteroduplex DNA molecules are created.molecules are created.

8. The recombination process is 8. The recombination process is repeated… repeated…

9. … until full-length double-9. … until full-length double-stranded heteroduplex DNA stranded heteroduplex DNA molecules are created. molecules are created.

10. The result is a library of 10. The result is a library of novel full-length genes which novel full-length genes which have different combinations of have different combinations of characteristics from the characteristics from the “parental” genes. “parental” genes.

etcetc… …

11. Test each recombinant for the 11. Test each recombinant for the desired property.desired property.