Dynamics of allele frequencies under different evolutionary and ecological scenarios.

<p>These diagrams indicate the proportion of alleles through time, with each color series representing those that arose from a common first mutation upon the ancestral (gray) genotype. A) The canonical model for adaptation in a single niche has been one of periodic selection, whereby beneficial mutations occur rarely enough that only one ever rises through the population at a time. B) Experimental evolution has repeatedly shown that many beneficial mutations can occur simultaneously and compete with each other before any one of them fixes, a scenario known as clonal interference. C) If multiple ecological niches exist, selection can drive a lineage to split into multiple, coexisting phenotypes (i.e., adaptive diversification). Lineages in each niche are indicated by either warm or cool colors and are separated by an orange dashed line representing the apparent equilibrium. Fixation events occur within each niche without eliminating diversity in the other niche. D) Both clonal interference and ecological diversification can operate simultaneously, giving rise to multiple lineages competing within each niche.</p

biologData

biologDat

growthRateData

Growth rate data for evolved and ancestral strain

Substrate dissimilarity does not predict metabolic erosion.

<p>(A) A simple categorization of substrates as sugars and nonsugars finds that the correlation between relatedness to glucose and evolved metabolic changes is the opposite from what is hypothesized. (B) The FBA-predicted mutational target size does not correlate with decreases in growth rate. (C) Hamming distance between FBA-generated flux vectors for carbon sources partially predicts ancestral growth rate. Black dots indicate the growth rate of the two ancestral strains. A total of 268 reactions were predicted as necessary for optimal metabolism on glucose. (D) Hamming distance between a substrate and glucose does not correlate with increases or decreases in growth rate. The <i>y</i> axis is the log of the ratio of growth rate relative to the ancestor, with all ratios greater or less than <i>e</i>² binned at the axis limit. For (C–D), purple dots are mutator strains, and orange dots are nonmutators. Larger dots at the axis extrema indicate more overlapping points, and the shading between purple and orange indicates the different proportions of mutators and nonmutators at that limit. For (B–D), substrates with the same <i>x</i> axis values were plotted with a slight offset, and the true value is listed in the axis label.</p

Relative growth rates across a variety of growth substrates for evolved strains from 20k (A) or 50k generations (B).

<p>Heatmaps indicate the log ratio of growth rates relative to the average of the two ancestors on that carbon source. White indicates a growth rate equal to that of the ancestor average, red faster, and blue slower. The growth rates are plotted on a log scale with the limits of the color range set for twice as fast and half as fast as the ancestor average. An “x” in a box indicates that no growth was observed for that combination of strain and substrate over 48 h. Strains that were mutators by that time point are indicated.</p

Biolog measurements are a poor proxy for growth performance.

<p>(A) Biolog AUC as measured for the D-glucose on Biolog plates. The evolved strains have a lower AUC value than the ancestor on glucose, the carbon source available during evolution (<i>p</i><0.0001, Welch's two sample <i>t</i> test). The mean AUC for the 20k and 50k isolates on glucose are not statistically different. (B) Scatter plot showing the measurement of function as Biolog AUC versus growth rate on all substrates, for all strains at 20k and 50k generations as well as the ancestors. The regression shown is for substrates after removal of categorical disagreements (growth without respiration or respiration without growth, 167/702 in total).</p

The methylotrophy specific metabolic network in in <i>M. extorquens</i> AM1.

<p>All genes, except for the <i>mau</i> cluster (gray), are present and >95% identical in <i>M. extorquens</i> PA1. <i>M. extorquens</i> AM1 and <i>M. extorquens</i> PA1 were grown on various C₁ and multi-C substrates (blue) for this study. Genes highlighted in red were deleted in <i>M. extorquens</i> PA1 to uncover that the metabolic network involved in methylotrophy in <i>M. extorquens</i> PA1 is identical to <i>M. extorquens</i> AM1. TCA: Tricarboxylic acid Cycle and EMC: Ethyl-malonyl CoA Pathway.</p

Acclimation and adaptation in an experimentally engineered and evolved bacterium.

<p>A) Combinations of acclimatizing and adaptive responses can be classified into four basic patterns based on wild-type (WT), perturbed (here, the engineered <i>Methylobacterium</i> strain, or “EM”), and evolved (EVO) physiological states. Physiological processes that were perturbed but return to a WT-like state are restored (blue); other processes that remain in a perturbed state are unrestored (red); those that are augmented from acclimation to adaptation are reinforced (orange); and still others are novel with respect to WT and EM states (green). B) Central one-carbon (C₁) metabolism of WT and EM strains. In EM, the native pathway of formaldehyde oxidation (grey box) has been disabled and replaced by a foreign plasmid expressing two genes – <i>flhA</i> and <i>fghA</i>, from <i>Paracoccus denitrificans</i> – whose protein products co-opt endogenous glutathione to generate a functionally analogous, yet non-homologous substitute for C₁ metabolism (blue box). This replacement results in the requirement for PntAB transhydrogenase to generate NADPH. C) EM was evolved in eight replicate cultures on methanol for over 600 generations. Isolates from each of the evolved populations (F1–F8) showed marked increases in growth rate and fitness relative to their EM ancestor. Line indicates y = x. D) Growth rates relative to EM on methanol are plotted for WT and the evolved isolates against two other C₁ compounds: methylamine and formate. Lines show linear regression with an r² of 0.94 and 0.73 for methylamine and formate, respectively, calculated in a Pearson correlation.</p

Temperature dependence of growth rate on alternative substrates.

<p>For all strain/substrate measurements, we determined the relative change in growth rate by changing temperature from 37°C to 30°C. For (A–B), purple dots are mutator strains; orange dots nonmutators. Points that fall outside of the plot range are plotted at the edge of the graph. (A) Effect of temperature change on 20k isolates. (B) Effect on 50k isolates. (C) For 50k isolates, the number of mutators and nonmutators that were rescued from no growth at 37°C to growth at 30°C.</p

Phenotypes of deletion mutants.

<p>(A) Schematic view of engineered deletion mutants. Each arc represents the deleted region in the mutant ET1 (dark blue), ET2 (green), ET3 (brown) and ET4 (grey). (B) Reaction norms of fitness for deletion mutants and wild type in 4 selective environments: M, S, MS, M/S, and each half-environment of M/S (M→S and S→M). (C) Transition time from S to M. (D) Fitness cost at stationary phase estimated as the fitness drop from hour 28 to hour 48. (E–G) Succinate-grown cultures with the following treatments: E, ampicillin (12.5 µg/mL); F, arsenate (30 mM); G, 36°C. Relative growth rate or final OD₆₀₀ (optical density) was calculated as the ratio of with and without treatment. Error bars represent 95% confidence intervals and significant differences from wild-type are indicated by *(P<0.05).</p