Evolution of Escherichia coli to 42 °C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations

Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering.