In Vivo Molecular Evolution Reveals Biophysical Origins of Organismal Fitness  Rafael Couñago, Stephen Chen, Yousif Shamoo  Molecular Cell  Volume 22, Issue 4, Pages 441-449 (May 2006) DOI: 10.1016/j.molcel.2006.04.012 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 In Vivo Continuous Evolution of Adenylate Kinase Produces Mutations that Are Varied in Side Chain Chemistry and Scattered throughout the Three-Dimensional Structure (A) Primary sequence alignment of B. subtilis and G. stearothermophilus AK showing positions and identities of mutations (colored arrowheads). Regions that are identical are indicated by asterisks. Four of the six mutations occur in regions that are conserved between the two proteins. Relevant domains for AK are indicated by black bars. (B) Mutations are mapped onto the structure of B. subtilis Q199R AK and shown as colored spheres. Mutations are found in both surface and buried regions of the protein. (C) Crystal structure of B. subtilis AK Q199R at 1.8 Å resolution showing the formation of a surface salt bridge that modestly stabilizes AK 1.7°C. Molecular Cell 2006 22, 441-449DOI: (10.1016/j.molcel.2006.04.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Frequency of AK Mutants in the Turbidostat as a Function of Temperature The adk gene was sequenced from 343 isolated strains. All 343 strains contained missense mutations to adk, and no silent mutations were observed. Strains were named according to their respective mutations to AK. After an initial 48 hr equilibration at 55°C, the temperature was increased 0.5°C/day from 55°C to 70°C. As temperature was increased in the turbidostat, samples were withdrawn and the cells plated at the temperature they were isolated. For this histogram, a total of 242 strains were isolated and sequenced. Q199R was the only mutant found from 55°C–61°C. At 62°C–63°C, five new double mutants are observed in the population as the Q199R strain begins to falter and moves toward extinction, and by 70°C, the double mutant Q199R/Q16L is dominant. Molecular Cell 2006 22, 441-449DOI: (10.1016/j.molcel.2006.04.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 All the Mutant Adenylate Kinases Isolated from Continuous Selection Exhibit Increases in Stability and Activity Maxima at Elevated Temperatures Compared to Wild-Type B. subtilis AK (A) Unfolding of mutant AKs was monitored by changes in circular dichroism spectra at 220 nm as a function of temperature. Different mutants are indicated by color and correspond to the data shown in Figure 2. The CD data suggest three broad categories of mutants: low temperature, wild-type and Q199R; intermediate temperature, Q199R/A193V, Q199R/T179I, Q199R/G213E, and Q199R/G214R; and high temperature, Q199R/Q16L. The three ranges of mutants correspond broadly to the rise and fall of mutants in the turbidostat. (B) Total activity at different temperatures was measured by separation of adenylate species by HPLC. At least two of the enzymes (Q199R/A193V and Q199R/T179I) show a significant increase in overall activity and provide a complementary strategy for improving organismal fitness by increasing enzyme activity as well as overall thermal stability. Means ± standard deviations for n = 2 are shown. Molecular Cell 2006 22, 441-449DOI: (10.1016/j.molcel.2006.04.012) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Fitness of the Two Major Double Mutants Q199R/A193V and Q199R/Q16L versus Q199R at Constant Temperature The fitness of each strain correlates with their success within the turbidostat. Selection coefficients (r) were determined from one-to-one competition at constant temperatures for mutants Q199R, Q199R/A193V, and Q199R/Q16L by serial transfer experiments. Strain Q199R/Q16L was the reference for all competition experiments. To estimate the relative fitness (W), the growth rate of the competitor was estimated by using the average Q199R/Q16L growth rate and the difference in selection rate. Molecular Cell 2006 22, 441-449DOI: (10.1016/j.molcel.2006.04.012) Copyright © 2006 Elsevier Inc. Terms and Conditions