ABSTRACT Title of Document: MECHANISMS OF RESISTANCE TO IONIZING RADIATION IN EXTREMOPHILES Kimberly Michelle Webb, Master of Science, 2012 Directed by: Dr. Frank T. Robb, Marine Estuarine Environmental Sciences Extremophiles display an astonishing array of adaptations to harsh environmental conditions. We analyzed the mechanisms of ionizing radiation resistance from a diverse group of extremophilic archaea and bacteria. In Halobacterium salinarum IR resistance is conferred by antioxidant Mn2+-complexes, and protein-free cell extracts (ultrafiltrates, UFs) of super-resistant (IR+) isolates of H. salinarum had increased concentrations of Mn, PO4 and amino acids compared to the founder strain. Proteomic analysis determined that IR+ isolates with increased Mn had elevated protein expression for central carbon metabolism, suggesting a Mn-stimulated metabolic route to increased IR resistance. We examined the role of mannosylglycerate, di-myo-inositol phosphate, and trehalose in the IR resistance of various thermophiles; aerobic thermophiles had UFs which were radioprotective of enzyme activity under aerobic conditions, which is attributed to Mn, PO4 and trehalose accumulation. In contrast, anaerobic thermophile UFs did not contain significant amounts of Mn, and were radioprotective only under anaerobic conditions; we conclude the anaerobic environment confers their IR resistance. MECHANISMS OF RESISTANCE TO IONIZING RADIATION IN EXTREMOPHILES By Kimberly Michelle Webb Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Master of Science 2012 Advisory Committee: Dr. Frank T. Robb, Chair Dr. Michael Daly Dr. Jocelyne DiRuggiero Dr. Kevin R. Sowers © Copyright by Kimberly Michelle Webb 2012 Dedication This work is dedicated to my entire family, especially my husband Nathan, and my parents Kevyn and Peter, for all your love and support. ii Acknowledgements I would like to thank Dr. Jocelyne DiRuggiero for her support, direction and patience throughout my graduate career. I would also like to thank various members of the DiRuggiero lab for their assistance and/or camaraderie: Shuhong Lu, Michelle Ploch, and especially Courtney Robinson. Thanks also to the members of my committee for their time and insight, Dr. Frank Robb, Dr. Kevin Sowers, and Dr. Michael Daly. I would like to thank Dr. Noboru Tomiya, for his insight and HPAEC analysis; Dr. Lai- Xi Wang and Jared Orwenyo (UMD School of Medicine) for their help with the compatible solute analysis; and Robert Cole (JHU School of Medicine) for his assistance with the proteomic analysis. I’d also like to acknowledge Jana Mihalic (JHU School of Public Health) for her ICP-MS and ion chromatography analysis, as well as for her morale-boosting discussion and accommodation for fast sample processing. A special thank you goes to Dr. Michael Daly and all the members of his lab at USUHS, Dr. Vera Matrosova, Dr. Elena Gaidamakova, and Margaret Wear, for their assistance and guidance at USUHS, access to the Co-60 irradiator, as well as their insight and thoughtful discussion. Lastly, thank you to my family and friends, especially my husband Nathan, and my parents; without your support none of this would have been possible. iii Table of Contents Dedication……………………….....………………………………………………….......ii Acknowledgements……………………………………………………………………....iii Table of Contents………,………………………………………………………………...iv List of Tables……………………………………………………………………..……….v List of Figures………………………………………………………………………..…...vi List of Abbreviations……………………………………………………………....……viii Chapter 1: Introduction……………………………………………………………...….....1 Ionizing Radiation Damage…………………………………………………………......4 Radioprotection and Damage Repair……………………………………………...….…7 Radiation Resistant Extremophiles…………………………………………………….15 Radiation Resistant Extremophile Systems…………………………………………....16 Research Objectives……………………………………………………………………23 Chapter 2: Paths to Extreme Resistance: Metabolic and Biochemical Factors Contributing to the Extreme Radiation-Resistant Mutants of H. salinarum Introduction……………………………………………………………………………29 Materials and Methods………………………………………………………………...30 Results………………………………………………………………………………....35 Discussion……………………………………………………………………………..57 Chapter 3: Radioprotection by Compatible Solutes and Mn2+ in Thermophilic Bacteria and Archaea Introduction…………………………………………………………………………….66 Materials and Methods………………………………………………………………....70 Results………………………………………………………………………………….75 Discussion……………………………………………………………………………...88 Chapter 4: Conclusions……………………………………………………………..........93 Appendix A…………………………………………………………………..…………..98 Appendix B......................................................................................................................103 Appendix C......................................................................................................................115 Appendix D......................................................................................................................116 References........................................................................................................................117 iv List of Tables Table 1-1. Examples of radiation resistant polyextremophiles………………………..16 Table 2-1. Examples of increased protein expression ratios unique to IR+ isolate 463...............................................................................................................55 Table 3-1. Ultrafiltrates and whole cell concentrations of Mn and Fe………………..78 Table 3-2. Ultrafiltrates and ethanol extracts concentrations for PO4, amino acids, and compatible solutes……………………………………………………………………. 78 v List of Figures Figure 1-1. Examples of extremophiles in the three domains of life……………………..3 Figure 1-2. Theoretical cellular reactions generating a variety of ROS following ionizing radiation (taken from [22])………………………………………………………6 Figure 1-3. Pathways for the repair of DNA double-strand breaks (from [30])………….9 Figure 1-4. Model for manganese-based chemical antioxidants in D. radiodurans…….13 Figure 1-5. Model of death by protein damage in irradiated cells………………………14 Figure 1-6. Compatible solutes in thermophiles………………………………………...18 Figure 1-7. Proposed pathways for superoxide detoxification in: aerobic and anaerobic organisms (from [30])…………………………………………………………………....21 Figure 2-1. Irradiation schedule and IR doses to obtain IR+ isolates of H. salinarum.....36 Figure 2-2. Survival of H. salinarum cultures exposed to 12 kGy of IR..........................37 Figure 2-3. Survival of H. salinarum isolates at 12 kGy of ionizing radiation................38 Figure 2-4. Survival curves of H. salinarum IR+ isolates and founder strain F3 exposed to 0, 8, 12, 15, and 18 kGy of ionizing radiation...............................................................39 Figure 2-5. Protection of enzyme activity........................................................................41 Figure 2-6. Ultrafiltrate composition for Founder 3 (F3) and the IR+ isolates 392, 393, and 463.............................................................................................................................43 Figure 2-7. Predicted proteome of H. salinarum vs. iTraq-identified proteins...............45 Figure 2-8. Classification of iTraq-identified proteins and predicted proteome of H. salinarum according to the CMR database...................................................................46 Figure 2-9. Ratios of protein expression levels between (A) F3-2 and F3-1, (B) 392-1 and F3-1, (C) 393-1 and F3-1, and (D) 463-1 and F3-1....................................................48 vi Figure 2-10. Decreased protein expression ratios (< 0.4) in IR+ isolates.........................51 Figure 2-11. Increased protein expression ratios (> 1.5) in IR+ isolates. .........................52 Figure 2-12. Classification of protein function for increased protein expression ratios among the three IR+ isolates..............................................................................................53 Figure 2-13. Classification of protein function for increased protein expression ratios for (A) Isolate 463, (B) Isolate 392, and (C) Isolate 393.........................................................56 Figure 3-1. Protection of enzyme activity.........................................................................79 2+ Figure 3-2. Protection of enzyme activity with trehalose, PO4 and Mn under aerobic conditions...........................................................................................................................81 2+ Figure 3-3. Protection of MG and DIP combined with PO4 and Mn under aerobic conditions...........................................................................................................................82 Figure 3-4. Protection of enzyme activity in aerobic and anaerobic conditions...............84 Figure 3-5. Protection of enzyme activity with compatible solutes..................................86 vii List of Abbreviations D10 – dose of ionizing radiation at which 10% of a microbial population survives DIP – di-myo-inositol phosphate DSB – double-strand break ESDSA - extended synthesis-dependent strand annealing H2O2 – hydrogen peroxide HO• – hydroxyl radical HR – homologous recombination IR – ionizing radiation IR+ – super-IR resistant MG – mannosylglycerate NHEJ – non-homologous end-joining •- O2 – superoxide PCR – polymerase chain reaction PER – protein expression ratio ROS – reactive oxygen