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Project Progress

This project uses experimental evolution to adapt lineages of the bacterium Escherichia coli to low temperature and examines the genetic basis and functional consequences of that adaptation. We previously developed a primary set of six clonal lineages and a secondary set of an additional 24 lineages adapted to 20°C. We are now in the process of developing twelve new lineages adapted to 14°C. After 800 generations, these 14°C lines have increased fitness over 15%, indicating a significant improvement in their ability to grow and compete at cold temperatures. Fitness improvement has been greatest in lines derived from the least cold adapted progenitors, indicating that the degree of prior cold adaptation is not necessarily a good predictor of future ability to adapt to cold conditions.

We have also evolved a set of 14 experimental populations of E. coli for 1000 generations under freeze-thaw-growth cycles. All have improved fitness in this regime owing to improved freeze-thaw survival and a shorter lag prior to the start of growth. To investigate the genetic changes responsible for this adaptation, six candidate genes were sequenced in clones from the evolved populations. However, no mutations were found relative to their progenitors. Next, we looked for genetic changes involving insertion sequence (IS) elements in several of the evolved populations, and a number of IS-associated changes were detected. Of particular interest, it was found that in six populations, an IS150 element inserted into the intergenic region of the uspA-uspB operon, which encodes Universal Stress Proteins A and B,. Also, in several populations, IS150 and IS186 elements were found to have inserted into cls, which encodes cardiolipin synthase. Goals now include determining the physiological significance of these mutations, and to test whether they are beneficial in freeze-thaw-growth conditions in isogenic strains.

As part of this project, we have purchased a set of PCR primers required to amplify every gene in E. coli and are using these to create our own custom-made genomic arrays. These will consist of a solid support onto which all 4,200 E. coli genes will be arrayed in a regular grid, allowing us to make genome-wide “queries” of gene expression and gene content of all of our cold-adapted lineages. In addition, these arrays will be made available to the research community.