Whole-genome sequencing analysis of two heat-evolved Escherichia coli strains.

BACKGROUND: High temperatures cause a suite of problems for cells, including protein unfolding and aggregation; increased membrane fluidity; and changes in DNA supercoiling, RNA stability, transcription and translation. Consequently, enhanced thermotolerance can evolve through an unknown number of genetic mechanisms even in the simple model bacterium Escherichia coli. To date, each E. coli study exploring this question resulted in a different set of mutations. To understand the changes that can arise when an organism evolves to grow at higher temperatures, we sequenced and analyzed two previously described E. coli strains, BM28 and BM28 ΔlysU, that have been laboratory adapted to the highest E. coli growth temperature reported to date. RESULTS: We found three large deletions in the BM28 and BM28 ΔlysU strains of 123, 15 and 8.5 kb in length and an expansion of IS10 elements. We found that BM28 and BM28 ΔlysU have considerably different genomes, suggesting that the BM28 culture that gave rise to BM28 and BM28 ΔlysU was a mixed population of genetically different cells. Consistent with published findings of high GroESL expression in BM28, we found that BM28 inexplicitly carries the groESL bearing plasmid pOF39 that was maintained simply by high-temperature selection pressure. We identified over 200 smaller insertions, deletions, single nucleotide polymorphisms and other mutations, including changes in master regulators such as the RNA polymerase and the transcriptional termination factor Rho. Importantly, this genome analysis demonstrates that the commonly cited findings that LysU plays a crucial role in thermotolerance and that GroESL hyper-expression is brought about by chromosomal mutations are based on a previous misinterpretation of the genotype of BM28. CONCLUSIONS: This whole-genome sequencing study describes genetically distinct mechanisms of thermotolerance evolution from those found in other heat-evolved E. coli strains. Studying adaptive laboratory evolution to heat in simple model organisms is important in the context of climate change. It is important to better understand genetic mechanisms of enhancing thermotolerance in bacteria and other organisms, both in terms of optimizing laboratory evolution methods for various organisms and in terms of potential genetic engineering of organisms most at risk or most important to our societies and ecosystems.