University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2002—pp. 85-86

George E. Fox (UH), Michael Travisano (UH), Robert Goldman (UH), and Fathi Karouia (UH)

Abstract
A primary goal of astrobiology is the understanding of the evolutionary and environmental limits of life. Radiation is a ubiquitous environmental challenge throughout the Universe. We are seeking to understand how changes in the radiation environment in the past or future may have effected or will effect evolutionary adaptation. Escherichia coli cell lines exposed to UV radiation were evolved for 60 days with a 25s daily UV B radiation exposure. We were able to achieve resistance to radiation, and 67 genes have initially been implicated in the response. The most notable were genes of the flagellar operon that were consistently up-regulated. Further studies are under way to understand the molecular basis of the adaptation.

TO SURVIVE OVER LONG PERIOD OF TIME, ORGANISMS CAN ASSUME forms that enable them to withstand extreme environments such as high-levels of radiation exposure, complete desiccation, and starvation. One of the main goals of the NASA astrobiology program¹ is to understand the evolutionary mechanisms and environmental limits of life. The goals also include exploring the biochemical and evolutionary strategies that push the limits of life by reinforcing, replacing, or repairing critical biomolecules and characterizing the structure and metabolic diversity of microbial communities in such extreme environments. This investigation will broaden our knowledge of an organism’s adaptation and will be critical for understanding how life might have established itself and survived in habitats beyond Earth.

In the past decade, the discipline has gained interest with several reviews published on extremophiles.^2-4 However, while much has been made of their physiological adaptations, the study of their evolution has largely been relegated to phylogenetics. Therefore, there is a need to understand the evolution of processes of extreme adaptation among organisms.

Earth’s unique ability to harbor life is attributed to the atmosphere which offers protection from the hazardous and harsh conditions of space. However, UV radiation has always played an important role in the evolutionary process of life. Furthermore, UV radiation is a ubiquitous selection pressure on Earth, and presumably, on other planets. The variations within organisms in their ability to tolerate UV on Earth suggest the operation of selective processes.

With the release of the complete E. coli genome sequence⁵ in 1997, this bacterium has maintained its position as the preferred model in biochemical genetics, molecular biology, and biotechnology. Recent advances in genomic technology have led to the development of high-throughput cDNA arrays, which measure, simultaneously, the expression levels of thousands of different genes.⁶

Results
The specific aim of this project was to evolve E. coli K12 for several hundred generations under a daily UV radiation exposure and determine how bacterial gene structure and regulation are affected. In order to address this goal, E. coli was grown under three conditions, (1) UV on LB plates, (2) no UV on LB plates, and (3) no UV in LB broth. Every day, for 60 days, the cells were exposed to 25 seconds of UV B and transferred to fresh medium. The bacterial gene expression was comprehensively examined using hybridization array assays. This effort employed Sigma-Genosys Panorama™ E. coli gene arrays, which contain 4,290 PCR-amplified open reading frames on a pair of duplicate nylon membranes. This represents all known protein-encoding genes in E. coli. Radioactive cDNA obtained from bacterial messenger RNA (mRNA) was used to probe the arrays. The experiment revealed the extent to which every gene in the cell was being transcribed.

Initial studies have indicated that after 600 generations cell lines exposed to a daily UV radiation achieve total resistance. More importantly, there was a four-fold increase in the survival rate between the selected lines and the ancestor. However, there appears to be a trade-off as the selected lines, although more competitively fit than the control lines, seem to grow more slowly. The expression of all genes in the UV exposed cell lines was compared to cell lines that had no UV exposure. Relative to the no UV control, a total of 23 genes were up-regulated and 44 were down-regulated (above three standard deviation and P < 0.05) in the UV adapted population. The most highly expressed up-regulated genes in absolute terms were all part of the flagellar operon suggesting that the ability to generate movement may be key to survival when the UV pulse occurs.

Future studies will emphasize the evaluation of the mutation rates of the selected cell lines and attempt to determine the mechanistic basis of resistance which should be correlated with the genomic comparison approach. As a part of these efforts, it is likely that we will re-sequence key regions of the genome to see what reactions occurred at the genetic level.

References
¹NASA Astrobiology Roadmap, final version, Nov. 20, 2002, accessed 2003. <http://astrobiology.arc.nasa.gov/roadmap>
²M. T. Madigan and B. L. Marrs. "Extremophiles," Sci. Am. 276 (1997): 82-87.
³K. Horikoshi and W. D. Grant. "Extremophiles," in Microbial Life in Extreme Environments. New York: Wiley-Liss, 1998.
⁴Journey to Diverse Microbial Worlds: Adaptation to Exotic Environments. Ed. J. Seekbach. Dordrecht: Kluwer Academic Publishers, 2000.
⁵F. R. Blattner, G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. "The Complete Genome Sequence of Escherichia coli K-12," Science 277.5331 (Sept. 5, 1997): 1453-74.
⁶C. S. Richmond, J. D. Glasner, R. Mau, H. Jin, and F. R. Blattner. "Genome-Wide Expression Profiling in Escherichia coli K-12," Nucleic Acids Res. 27.19 (1999): 3821-35.

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