Experimental Molecular Evolution Evolution of bacterial resistance to

Experimental Molecular Evolution

Evolution of bacterial resistance to antibiotics D. M. Weinreich, N. F. Delaney, M. A. De. Pristo & 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 (A 42 G, E 104 K, M 182 T, G 238 S) and one 5' noncoding mutation (g 4205 a). 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 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 Thermotogale Thermus Actinobacteria Cyanobacteria Bacillus Green Sulfur Spirochaete Green Sulfur Cyanobacteria Thermus Spirochaete Proteobacteria

Maximum Likelihood Tree ML-Stem Alternative Tree Outgroup Thermotogale Thermus Alt-Stem Actinobacteria Cyanobacteria Bacillus Green Sulfur Spirochaete Green Sulfur Cyanobacteria Thermus Spirochaete Proteobacteria

Maximum Likelihood Tree ML-Stem Alternative Tree Outgroup Thermotogale Thermus Alt-Stem Actinobacteria Cyanobacteria ML-Meso Bacillus Green Sulfur Spirochaete Green Sulfur Cyanobacteria Thermus Spirochaete 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, ER 2566), induced with IPTG This results in the translation of a fusion construct containing: - Chitin Binding Domain - Intein - EFTu gene

EF-Tu Antibody 111 k. Da 66 k. Da 45 k. Da Precursor CBD-Intein EF-Tu

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.