Technological advances in analytical flow cytometry and cell sorting are rapidly revolutionizing our understanding of microbial genetic diversity and its relationship to function in widely varied environmental systems. The in situ application of flow cytometry and high-speed sorting is now an indispensable component for interrogation of the physiological state and activity of subpopulations and assessment of the importance of specific genes to cell function. We propose to apply emerging techniques in analytical flow cytometry, high-speed cell sorting, and single-cell multiple displacement amplification (MDA) to Antarctic ice samples ranging 100 Kyr to 8 Myr in order to assess the relative proportion of viable vs. dead cells and to explore genetic strategies of DNA repair in ancient ice microbes. Recovery of viable microbes within ice exposed to cosmic radiation for ~8 Myr suggests that subpopulations of microbes within ancient ice possess particularly efficient DNA repair mechanisms. Given the strong influence of cosmic flux on DNA degradation in ancient ice samples and evidence of effective DNA repair, we hypothesize that the degree of DNA damage increases with time and that viable 8 Myr old bacteria possess particularly effective and novel DNA repair mechanisms for which we can obtain genetic signatures. The research goal is to obtain a library of genes used for DNA repair by viable bacteria from the oldest ice on earth. Using analytical techniques not previously applied to ancient ice microbes. Thus, the proposed research is an 'exploratory' endeavor with potentially high risk. It is likely that novel genetic capabilities will be discovered through our analyses, perhaps providing key molecular insight into the survival of life over geological time, and perhaps on other planets. Given the use of standard flow cytometry instrumentation and the increasing affordability of genome analyses, our research strategy is transferable to the scientific community and should offer key advantages to the study of ice microbes. This project will allow for continued professional development of a young PI and provide an opportunity whereby researchers with different educational backgrounds (undergraduate students, graduate students, technician, post-docs, and faculty) can interact and develop.
