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 wild-type 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.

Fig 1

Graphical depiction of adaptation process. The top panel shows the temperature of the Evolugator culture chamber as a function of time (days) for a short 12-day window of the experiment, which lasted a total of 234 days. The bottom panel shows the optical density of the culture in the growth chamber during the same time period. Two examples of heat shock are shown in this time period. During these heat shocks, the temperature is increased to stress the cells. There is a subsequent decrease in the optical density of the cultures due to heat shock. The temperature is then rapidly decreased, allowing the cultures to recover and optical density to increase again. Temperature is then increased to select for mutants that might have arisen that are more thermotolerant.

Fig 2

Thermotolerance of wild type, EVG1064, and EVG1058. (A) Growth of MG1655 and EVG1064 E. coli strains on LB plates at 30°C, 37°C, and 48.5°C; (B) mean generation times of MG1655 and EVG1064 in liquid LB medium plotted as a function of temperature; (C) final cell densities, as measured by OD₆₀₀, at 24 hours for MG1655 and EVG1064 grown in liquid LB medium at various temperatures; (D) resistance to 30-min exposures to elevated temperatures for MG1655 and EVG1064 as measured by growth on LB plates; (E) mean generation times of EVG1058 and EVG1064 in liquid LB medium plotted as a function of temperature. For all panels, error bars indicate ±1 standard deviation. For panels B and E, asterisks denote temperatures at which the growth rates of the two compared strains are significantly different using a 2-tailed, type 2 t test. **, P < 0.005; *, P < 0.05.

Fig 3

Pulsed-field gel electrophoresis of XbaI-digested genomic DNA from MG1655 and EVG1064. Lanes: 1, lambda ladder; 2, MG1655; 3, EVG1064; 4, low-range ladder; 5, midrange ladder; 6, MG1655; 7, EVG1064; 8, lambda ladder. Notice the lack of difference between the fragmentation patters between MG1655 and EVG1064, which is indicative of the absence of large chromosomal rearrangements between the two strains.

Fig 4

Phenotypic analysis of glpF and fabA. All growth curves were obtained in liquid LB medium. (A) Mean generation times of wild-type BW25113 and its corresponding glpF deletion mutant (ΔglpF mutant) plotted as a function of temperature; (B) mean generation times of EVG1064 carrying either the glpF or folE expression plasmids as a function of temperature; (C) mean generation times of EVG1064 carrying either the fabA or folE expression plasmids as a function of temperature. For all panels, error bars indicate ±1 standard deviation. Asterisks denote temperatures at which the growth rates of the two strains depicted are significantly different using a 2-tailed, type 2 t test. **, P < 0.005; *, P < 0.05. For panels B and C, 24 μg/ml chloramphenicol is added to the LB to select for plasmid retention.