Adaptive laboratory evolution of a thermophile toward a reduced growth temperature optimum.

Thermophily is an ancient trait among microorganisms. The molecular principles to sustain high temperatures, however, are often described as , somewhat implying that they evolved from a non-thermophilic background and that thermophiles, i.e., organisms with growth temperature optima (T) above 45°C, evolved from mesophilic organisms (T 25-45°C). On the contrary, it has also been argued that LUCA, the last universal common ancestor of and , may have been a thermophile, and mesophily is the derived trait. In this study, we took an experimental approach toward the evolution of a mesophile from a thermophile. We selected the acetogenic bacterium (T 66°C) since acetogenesis is considered ancient physiology and cultivated it at suboptimal low temperatures. We found that the lowest possible growth temperature (T) under the chosen conditions was 39°C. The bacterium was subsequently subjected to adaptive laboratory evolution (ALE) by serial transfer at 45°C. Interestingly, after 67 transfers (approximately 180 generations), the adapted strain Adpt45_67 did not grow better at 45°C, but a shift in the T to 60°C was observed. Growth at 45°C was accompanied by a change in the morphology as shorter, thicker cells were observed that partially occurred in chains. While the proportion of short-chain fatty acids increased at 50°C vs. 66°C in both strains, Adpt45_67 also showed a significantly increased proportion of plasmalogens. The genome analysis revealed 67 SNPs compared to the type strain, among these mutations in transcriptional regulators and in the cAMP binding protein. Ultimately, the molecular basis of the adaptation of to a lower T remains to be elucidated. The observed change in phenotype is the first experimental step toward the evolution of thermophiles growing at colder temperatures and toward a better understanding of the cold adaptation of thermophiles on early Earth.