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Project Progress

This project uses experimental evolution to adapt lineages of the bacterium Escherichia coli to low temperature and then examines the genetic basis and functional consequences of that adaptation. It includes two main projects, one of which addresses adaptation to growth at progressively lower temperatures while the other examines adaptation to alternating freeze-thaw-growth conditions.

Recent efforts on the first project include the development of 12 new lineages adapted to 14°C which were derived from 4 different ancestors. These new lineages, which have undergone over 1800 generations, exhibit an increased competitive fitness of 22% on average. However, there is highly significant heterogeneity among the lines derived from the different progenitors. Two of the progenitors have produced lines with about a 40% on average increase in relative fitness; another produced lines with an increased fitness of only about 7%; and lines from a fourth progenitor have not shown any significant improvement of fitness for the 14°C regime. These data indicate that subtle differences in the starting genotype, even within the same species, can strongly constrain subsequent adaptation to low temperature. After the evolution experiment has run for 2000 generations, we will undertake further analysis of the evolutionary changes in fitness at 14°C as well as possible trade-offs at other temperatures, thermal niche limits and breadth, and underlying genomic changes that are responsible for this cold adaptation.

In the second project, we evolved a set of 14 experimental populations of E. coli for 1000 generations under freeze-thaw-growth cycles. All populations achieved greater fitness under this regime owing to improvements in two aspects of their performance: greater survival during freeze-thaw cycles, and a shorter lag phase after they thaw that allows the bacteria to commence growth sooner. Multiple approaches are being taken to determine the genetic changes responsible for these adaptations. In one, we screened the entire genome of several evolved lines for changes that involve insertion sequence (IS) elements and found, in 8 independent populations, IS150 elements inserted into the uspA-uspB intergenic region encoding universal stress proteins A and B. These results strongly implicate this locus as having a role in the improved freezing tolerance phenotype.

We also found that in several of the populations, IS150 and IS186 elements had inserted into cls, which encodes cardiolipin synthase. IS insertion mutations are often difficult to manipulate, but a more tractable 11-bp deletion mutation was also found in the cls gene in another population. This mutation has served as a focus for understanding the effects of cls mutations on cell physiology and performance in the freeze-thaw-growth regime. To that end, we reverted the cls deletion back to its ancestral state in the relevant freeze-thaw-growth line in order to produce two clones that are isogenic except for the cls gene. Reversion of this evolved mutation reduced fitness by about 25% under the freeze-thaw-growth regime, which means that the cls deletion mutation is highly beneficial in that environment. The effect of this beneficial mutation appears to improve freeze-thaw survival more than it affects subsequent growth. Furthermore, the beneficial affect of this mutation is specific to the freeze-thaw-growth regime, as there is no measurable benefit under growth-only conditions or after a cold shock. Experiments are now underway to measure the effects of cls mutations on membrane fluidity, while future work will seek to determine the physiological significance of the uspA-uspB mutations.