Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Abstract

Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/ maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wildtype fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

“…1). Prior to this study, the highest growth temperature for E. coli cells was reported to be around 48.5°C, as achieved by experimental evolution (5,16). Here, we found that the CeHSP17 protein is, at least partially, localized at the E. coli cell envelope (Fig.…”

Specificity of genome evolution in experimental populations of Escherichia coli evolved at different temperatures

“…For example, increased chaperonin expression (47), reduced glycerol transport (48), and altered RNA polymerase activity (46,49) have been found in E. coli strains selected for increased growth at 42°C or for extending the upper limit on growth to as high as 48.5°C. Single mutations are often responsible for most of the improvements in any one evolved strain (48,49), although this pattern would presumably not hold for experiments of longer duration. Some studies have found that E. coli evolved at stressfully high temperature tend to restore their overall gene expression profiles toward the baseline levels corresponding to their ancestor when it is grown the optimal growth temperature of 37°C (49,50).…”

Laboratory Evolution of Microbial Interactions in Bacterial Biofilms

“…Lenski's experiment is still in progress, reaching 64,000 generations in January 2016, and many similar studies have been performed by other researchers on different microbial species (not only bacteria but also yeasts, phages, or protists) covering basic scientific questions (29,30) or even in directly applicative projects (31). While planktonic cultures offer a simple setup to study the evolution of bacteria (23,28,31,32), several studies have shown that the selective pressures in unstructured conditions differ from those in biofilms (33)(34)(35)(36). For example, homogeneous, well-mixed environments may select against "social" genotypes that secrete costly metabolites, so-called "public goods," and rather favor fast-reproducing selfish individuals (37).…”