Metabolic Erosion Primarily Through Mutation Accumulation, and Not Tradeoffs, Drives Limited Evolution of Substrate Specificity in Escherichia coli

Evolutionary adaptation to a constant environment is often accompanied by specialization and a reduction of fitness in other environments. We assayed the ability of the Lenski Escherichia coli populations to grow on a range of carbon sources after 50,000 generations of adaptation on glucose. Using direct measurements of growth rates, we demonstrated that declines in performance were much less widespread than suggested by previous results from Biolog assays of cellular respiration. Surprisingly, there were many performance increases on a variety of substrates. In addition to the now famous example of citrate, we observed several other novel gains of function for organic acids that the ancestral strain only marginally utilized. Quantitative growth data also showed that strains with a higher mutation rate exhibited significantly more declines, suggesting that most metabolic erosion was driven by mutation accumulation and not by physiological tradeoffs. These reductions in growth by mutator strains were ameliorated by growth at lower temperature, consistent with the hypothesis that this metabolic erosion is largely caused by destabilizing mutations to the associated enzymes. We further hypothesized that reductions in growth rate would be greatest for substrates used most differently from glucose, and we used flux balance analysis to formulate this question quantitatively. To our surprise, we found no significant relationship between decreases in growth and dissimilarity to glucose metabolism. Taken as a whole, these data suggest that in a single resource environment, specialization does not mainly result as an inevitable consequence of adaptive tradeoffs, but rather due to the gradual accumulation of disabling mutations in unused portions of the genome. Author Summary Adaptation to a single constant environment is commonly expected to result in decreased performance in alternative conditions, or specialization. It has been proposed that, rather than occurring through the neutral accumulation of mutations in unused alternative pathways, this happens because loss of these pathways enhances fitness in the constant environment via tradeoffs. We examined growth rates across a variety of nutrients for 12 independent lineages of Escherichia coli that had evolved in the laboratory for decades in a glucose-containing medium. Surprisingly, after 20,000 generations there were actually widespread improvements in the use of alternative nutrients, rather than the expected declines. After 50,000 generations, however, we find that this trend reversed for those populations that evolved a much higher mutation rate. This indicates that high mutation rate, and not adaptive tradeoffs per se (as had been previously proposed), is the primary driver of specialization. These results caution against general assumptions about the importance of adaptive tradeoffs during evolution, and emphasize the key role that newly evolved changes in mutation rate can play in promoting niche specialization.