Experimental Evolution of UV-C Radiation Tolerance: Emergence of Adaptive and Non-Adaptive Traits in Escherichia coli Under Differing Flux Regimes

Tuesday, 16 December 2014: 5:30 PM
Amy Moffet1, Alexis Okansinski2, Camilla Sloan3, Joseph M Grace4, Ivan G Paulino-Lima5, Diana Gentry6, Lynn J Rothschild6 and Manel Camps7, (1)University of California Santa Cruz, Biomolecular Engineering, Santa Cruz, CA, United States, (2)College of San Mateo, San Mateo, CA, United States, (3)Cañada College, Redwood City, CA, United States, (4)NASA Ames Research Center, Education Associates Program, Moffett Field, CA, United States, (5)NASA Ames Research Center, Oak Ridge Associated Universities, Moffett Field, CA, United States, (6)NASA Ames Research Center, Biospheric Science Branch, Moffett Field, CA, United States, (7)University of California Santa Cruz, Microbiology & Environmental Toxicology, Santa Cruz, CA, United States
High-energy ultraviolet (UV-C) radiation is a significant challenge to life in environments such as high altitude areas, the early Earth, the Martian surface, and space. As UV-C exposure is both a selection pressure and a mutagen, adaptation dynamics in such environments include a high rate of change in both tolerance-related and non-tolerance-related genes, as well changes in linkages between the resulting traits. Determining the relationship between the intensity and duration of the UV-C exposure, mutation rate, and emergence of UV-C resistance will inform our understanding of both the emergence of radiation-related extremophily in natural environments and the optimal strategies for generating artificial extremophiles.

In this study, we iteratively exposed an Escherichia colistrain to UV-C radiation of two different fluxes, 3.3 J/m^2/s for 6 seconds and 0.5 J/m^2/s for 40 seconds, with the same overall fluence of 20 J/m^2. After each iteration, cells from each exposure regime were assayed for increased UV-C tolerance as an adaptive trait. The exposed cells carried a plasmid bearing a TEM beta-lactamase gene, which in the absence of antibiotic treatment is a neutral reporter for mutagenesis. Sequencing of this gene allowed us to determine the baseline mutation frequency for each flux. As an additional readout for adaptation, the presence of extended-spectrum beta-lactamase mutations was tested by plating UV-exposed cultures in cefotaxime plates.

We observed an increase of approximately one-million-fold in UV-C tolerance over seven iterations; no significant difference between the two fluxes was found. Future work will focus on identifying the genomic changes responsible for the change in UV-C tolerance; determining the mechanisms of the emerged UV-C tolerance; and performing competition experiments between the iteration strains to quantify fitness tradeoffs resulting from UV-C adaptation.