Evolution after introduction of a novel metabolic pathway consistently leads to restoration of wild-type physiology

Abstract

Organisms cope with physiological stressors through acclimatizing mechanisms in the short-term and adaptive mechanisms over evolutionary timescales. During adaptation to an environmental or genetic perturbation, beneficial mutations can generate numerous physiological changes: some will be novel with respect to prior physiological states, while others might either restore acclimatizing responses to a wild-type state, reinforce them further, or leave them unchanged. We examined the interplay of acclimatizing and adaptive responses at the level of global gene expression in Methylobacterium extorquens AM1 engineered with a novel central metabolism. Replacing central metabolism with a distinct, foreign pathway resulted in much slower growth than wild-type. After 600 generations of adaptation, however, eight replicate populations founded from this engineered ancestor had improved up to 2.5-fold. A comparison of global gene expression in wild-type, engineered, and all eight evolved strains revealed that the vast majority of changes during physiological adaptation effectively restored acclimatizing processes to wild-type expression states. On average, 93% of expression perturbations from the engineered strain were restored, with 70% of these occurring in perfect parallel across all eight replicate populations. Novel changes were common but typically restricted to one or a few lineages, and reinforcing changes were quite rare. Despite this, cases in which expression was novel or reinforced in parallel were enriched for loci harboring beneficial mutations. One case of parallel, reinforced changes was the pntAB transhydrogenase that uses NADH to reduce NADP(+) to NADPH. We show that PntAB activity was highly correlated with the restoration of NAD(H) and NADP(H) pools perturbed in the engineered strain to wild-type levels, and with improved growth. These results suggest that much of the evolved response to genetic perturbation was a consequence rather than a cause of adaptation and that physiology avoided "reinventing the wheel" by restoring acclimatizing processes to the pre-stressed state.

Figure 1. Acclimation and adaptation in an experimentally engineered and evolved bacterium.

A) Combinations of acclimatizing and adaptive responses can be classified into four basic patterns based on wild-type (WT), perturbed (here, the engineered Methylobacterium 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 – flhA and fghA, from Paracoccus denitrificans – 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.

Figure 2. Microarray analysis of changes in gene expression in WT, EM, and each of the evolved strains.

A) Genes with significant differences in expression in WT or the F isolates relative to EM. Each gene has a single value for the log₂ difference in expression of WT relative to EM, and up to eight different values for each evolved strain. Changes in expression were categorized and colored as in Figure 1A as restored (blue), unrestored (red), reinforced (orange), or novel (green), as well as a fifth class of “partially restored” expression (purple). The histogram (right) bins each of these observations only considering adaptation, and thus just the differences between EM and the evolved strains. B) Instances of restored, unrestored, partially restored, reinforced, or novel expression across each of the eight evolved strains. C) Principal component analysis of all differentially expressed genes from physiological acclimation and adaptation.

Figure 3. Parallelism of gene expression changes across categories.

Instances of fully (blue) and partially (purple) restored, unrestored (red), reinforced (orange), or novel (green) changes in gene expression that occurred in parallel across the evolved lines. Novel is separated here into both increases (light green) and decreases (dark green) in expression for that gene. Dashed lines represent the parallelism of partially restored genes when combined with either fully restored (dashed blue) or fully unrestored (dashed red) changes.

Figure 4. Multiple evolved mechanisms reinforce increased pntAB expression and transhydrogenase activity.

A) Known (F4) and candidate (F3) mutations that increase pntAB expression. B) Relative increases in pntAB expression were highly correlated with increased overall transhydrogenase activity (Pearson correlation, r² = 0.96 with p = 1.27×10⁻⁵). C) Plot of increasing transhydrogenase activity with increasing growth rate. WT is able to grow well in the absence of transhydrogenase; however, enzyme activity is significantly increased from WT to EM, and reinforced even further from EM to each of the evolved lineages (p<0.05, Welch two-sample t-test). A significant positive relationship exists between transhydrogenase activity and growth rate for the evolved isolates (Pearson correlation, r² = 0.86 with p<0.01).

Figure 5. The redox states of pyridine nucleotides are perturbed in EM but restored through evolution.

The relative ratios of NADPH/NADP⁺ (A) and NADH/NAD⁺ (B) plotted against growth rate for WT, EM, and each of the evolved strains. The redox states of NADP(H) and NAD(H) were perturbed in EM but returned toward WT-like values in almost all of the evolved lineages. Ratios were highly correlated with growth rate for both NADP(H) (r² = −0.88 with p = 6.9×10⁻⁴) and NAD(H) (r² = −0.76 with p = 0.011) in a Pearson correlation.