Deinococcus Launches Aboard Endeavor’s Last Flight

1. Deinococcus Launches Aboard Endeavor’s Last Flight The four life forms flown in Shuttle LIFE were Tardigrades; the bacteria Deinococcus radiodurans and Bacillus subtilis; and the archaeon Pyrococcus furiosus. A passenger manifest explained what characteristics of the different microorganisms - such as resistance to radiation, and extreme hardiness - made them good choices for space travel. May, 2011

2. MCB Seminar April 25, 2013 3:30 pm - 4:30 pm Lecture Room C USUHS Radiation- Resistant Deinococcus radiodurans Deinococcus Mn2+ Complexes: New FrontiersMichael J. DalyUniformed Services University of the Health Sciences (USUHS)Bethesda, MD 20814, USAEmail: michael.daly@usuhs.edu Deinococcus History The Paradox Mn2+ Antioxidants Protein Protection Applications

3. Ongoing Fukushima Disaster Reminds the World that Research on Prevention of Acute and Chronic Radiation Effects is Critical “Perhaps if we knew why Deinococcus cells are so resistant to radiation, we could find ways to protect people from atomic radiation”. - A new perspective on radiation resistance.Nature March 2011: Reactors # 1, 3, 4 Leaking Cold War Radioactive Waste Catastrophic Fukushima Nuclear Accident April 8, 2013: Another major radioactive tank leak discovered Level 7 March 10, 2013: Major radioactive tank leak discovered Nuclear Threats Burns from radiation devices known since Thomas Edison’s public displays of x-rays in 1896 March 26, 2013: North Korea aims nuclear missile at US

4. Extreme Ionizing Radiation Resistance Since 1956, 42 distinct species of Deinococcus reported 3 days, 25oC, 60 Gy/hour on nutrient agar 137Cs 60 Gy/hour D. roseus Antarctica Japan D. grandis D. radiopugnans Nottingham Peru D. proteolyticus Portugal N. Atlantic D. geothermalis D. radiophilus

5. 0.5 mm http://www.usuhs.mil/pat/deinococcus/index_20.htm

6. Phylogenetic distribution of radiation resistant organisms. The existence of so many unrelated radioresistant species suggests that the molecular mechanisms that protect against ionizing radiation-induced damage evolved independently in these organisms. Evolving high-level radiation resistance is not so difficult, as demonstrated in the lab with various bacteria including E. coli

7. The Model complexes Haber-Weiss Reaction ROS Production under Aqueous Conditions by Ionizing Radiation (x-Rays & g-Rays)

8. 1960-2004: As radiation was deemed to damage cellular macromolecules indiscriminately, and as genes exist at far lower abundance in cells than their products, genes assumed the role of the most important targets – early on, the DNA double strand break (DSB) was identified as the critical lesion. And, DNA DSB repair mutants seemed to confirm this – all such mutants were highly radiation-sensitive. DNA Repair (Amst). 2012 Jan 2;11(1):12-21: DSB Yields for g-Rays 0.2 DSB/Gy/Mbp 0.0005 DSB/Gy/Mbp 0.05 DSB/Gy/Mbp 0.005 DSB/Gy/Mbp In all cell types tested so far – mammalian cells, simple eukaryotes, archaea, bacteria – DSB lesion yields for ionizing radiation are essentially the same: ~0.005 DSB/Gy/Mbp

9. Survival Curves and DSBs 100% Representative Insects Rotifers C. elegans Fungi Halobacteria Cyanobacteria Deinococci Amoebae Deinococcus radiodurans Caenorhabditis elegans Escherichia coli Survival Human lung fibroblasts Shewanella oneidensis You and most of Life g-rays 5 DSBs 400 DSBs 120 DSBs 72 DSBs <1 DSB 10% g-Rays Dose/Gy 0 1 10 100 1000 10000 UVC Dose/J/m2 0 0.1 1 10 100 1000 Desiccation Dose/days 0 0.1 1 10 100 1000

10. So, the central question became: What is the molecular basis of extremely efficient DSB repair in Deinococcus? nucleoids genes Mars Mn Pigments The New York Times (1999) The D. radiodurans genome does not appear to encode an unusual set of DNA repair genes that is distinct from those in radiation-sensitive bacteria.

11. For a given dose of g-radiation, the level of DNA damage in D. radiodurans compared to all other organisms is very similar ~0.004 DSB/Gy/Haploid Genome D. radiodurans 17,500 Gy 20 kb The Paradox D. radiodurans Post-17,500 Gy 24 hours later

12. In 2004, we reported that Mn accumulation closely linked to Deinococcus radiation resistance. But, Mn didn’t protect DNA So, What is Mn protecting? ● Deinococcus hoards Mn2+ (0.25-1 mM) in cytoplasm, but Fe out. ● As resistance in different bacteria went up, so did their [Mn] Mn2+ Fe

13. Yet, for a given dose of g-radiation, the level of protein damage in Deinococcus compared to other bacteria is very different Resistance Fe The Bacteria Mn Exposed to Same Dose The Paradox Same Protein Purification Procedure Protein Oxidation Assay Protein Carbonyl A founding concept of radiobiology that deals with X-rays + g-rays is that radiation indiscriminately damages cellular macromolecules. Whereas DNA lesion-yields in cells exposed to a given dose radiation are fixed, protein lesion-yields are highly variable and closely related to survival.

14. The Model Protein Damage complexes Haber-Weiss Reaction

15. But, the nature of the radioprotective agents and their targets remained a mystery for 40 years!

16. Something in D. radiodurans protein-free ultrafiltrates protects proteins Mn2+ In vitro + EC proteins + Ultraftr. g-radiation ! + ! g-radiation BamHI ! + BamHI desiccation

17. Approach to Isolating Protective Mn2+ Complexes in Extremely Radiation Resistant Organisms Protein-Free Extracts Small-Molecule Analysis Ultrafiltrates Ultracentrifugation + Ultrafiltration What is enriched? Mn(II) Orthophosphate Peptides Nucleosides HPLC MS - Metabolomics Chromatography Atomic Abs Spec Reconstituted Complexes Enzyme radiation activity assays. Carbonyl assays. Radioprotection of human cells and bacteria. Applications Radioprotection Enzyme storage Vaccine preparation In vitro and in vivo screening

18. Composition of theDR-ultrafiltrate Mn complexes Free amino acids and peptide-derived amino acids d

19. Putative D. radiodurans Mn2+ Complexes Orthophosphate Nucleoside- analogs O2·- O2·- HO· Amino acids/ Peptides

20. Using various forms of paramagnetic spectroscopy: Professor Brian Hoffman Department of Chemistry Northwestern University 2145 Sheridan Road Evanston, IL 60208-3113 Ajay Sharma, Elena K. Gaidamakova, Vera Y. Matrosova, Brian Bennett, Michael J. Daly, Brian M. Hoffman. Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to γ-radiation by advanced paramagnetic resonance methods. Proc. Natl. Acad. Sci. USA March, 2013 In cells of D. radiodurans, Mn2+:E.coli S.cerevisiae (Sensitive) (Moderately resistant) ● is bound to orthophosphate not polyphosphate +++ +++ ● is bound to water (+) +++++ ● mainly bound to small molecules (SM) not enzymes enzymesSM ● coordinated with N (but probably also C=O) - - - - - - ● coordination is not altered by megadoses of g-rays altered!altered a bit

21. Extraordinary Antioxidant Synergism (BamHI Assay) For example: 25 mM 25 mM PiB 3 mM U 1mM MnCl2 25 mM PiB 25 mM PiB 1mM MnCl2 3 mM U 25 mM 3 mM U 1mM MnCl2 + BamHI + BamHI + BamHI + BamHI + BamHI + Then, BamHI + l Mg2+, Ca2+, Zn2+, Ni2+, Cu2+ and Fe2+ have no protective effect when combined with uridine (U) and PiB (phosphate buffer, pH 7.4)

22. Glutamine Synthetase !!! We were Stunned! Aqueous Glutamine Synthetase (GS) Mixture: D20 GS activity/ g-ray dose: 25 mM PPB (pH7.4): 150 Gy 25 mM PPB (pH7.4) + 1 mM Mn: 1,800 Gy 25 mM PPB (pH7.4) + 1 mM Mn + 10 mM Leu: >15,000 Gy 25 mM PPB (pH7.4) + 1 mM Mn + 3 mM (A+U): >25,000 Gy 25 mM PPB (pH7.4) + 1 mM Mn + 3 mM Decapeptide:>40,000 Gy New Designer “Deinococcus Peptides (NIH) + Mn +Pi >100,000 Gy!!

24. Reminder: Survival Curves and DSBs 100% Representative Insects Rotifers C. elegans Fungi Halobacteria Cyanobacteria Deinococci Amoebae Deinococcus radiodurans Caenorhabditis elegans Escherichia coli Survival Human lung fibroblasts Shewanella oneidensis You and most of Life 5 DSBs 400 DSBs 120 DSBs 72 DSBs <1 DSB 10% g-rays Dose/Gy 0 1 10 100 1000 10000 UVC Dose/J/m2 0 0.1 1 10 100 1000 Desiccation Dose/days 0 0.1 1 10 100 1000

25. From Worms to Bacteria: DSB Repair Efficiencies Depend on Protein Protection

26. Application

27. Lambda Phage + DR Mn-Complexes (Mn-pep-Pi) tail tail DNA is destroyed 40,000 Gy + Mn-pep-Pi 40,000 Gy Proteins survive Immunogenic! Everything is wiped out DNA Destroyed Protein & Structure Survives! All USUHS Irradiated Vaccine (See next slide for NIH study) Next Slide – Details of Collaboration between Daly and Datta Groups

28. MRSA MRSA

29. VEE VIRUSES

30. Ex Vivo Radioprotective Effects of Reconstituted D. radiodurans Mn-Peptide Complexes on Human Cells 100% Cell Viability % 0% 100 0 Radiation Dose (Gy) Reconstituted Complex DR-ultrafiltrate Control Collaboration between Daly and Tom Lamkin’s Group

31. Mn-Complexes applied to the growth medium of E. coli endows it with extreme radiation resistance under high-level chronic gamma irradiation The “concentric ring-images” on the agar plates below were developed under high-level chronic Cs-137 radiation using E. coli growth to show where the radioprotective Mn complexes were applied. E. coli No Radiation + 42 Gy/hour D.radiodurans D. radiodurans E. coli + 42 Gy/hour + 42 Gy/hour

32. Take-Home Messages: ● At least in prokaryotes, protein oxidation in irradiated cells is not the consequence of cell death, but its major probable cause – If you want to survive radiation and other forms of oxidative stress, protect your proteins! ● A direct route to extreme radiation resistance appears to be by metabolic regulation, ie, via metabolite accumulation, which protects proteins from ROS. ●The possibility that Mn-dependent chemical antioxidants in D. radiodurans are based on common metabolites raises the possibility that equivalent synergistic processes promoted by Mn 2+ may be acting similarly in other organisms, and perhaps also in mitochondria and their mammalian hosts. ●Practical areas which are impacted: 1. bioremediation of high-level radioactive waste sites; 2. long-duration enzyme/antibody storage; 3. metabolic interventions at the cellular level which mitigate oxidative stress during irradiation or aging; and 4. vaccine preparation.

33. Latest information: : Daly + Deinococcus Daly et al., Science306, 925-1084 (2004) Daly et al., PLoS Biology, 5(4) (2007) Daly et al., ISME J, 2, 393-403 (2008) Daly, Nat. Rev. Microbiol.,7, 237-45 (2009) Daly et al., PLoS One., e2349j (2010) Daly, DNA Repair 11, 12-21 (2011) Gaidamakova et al., Cell H-M,12, (2012) Daly and Culotta, Antioxidant&Redox Sig (2012) Hoffman and Daly et al. PNAS March (2013) Big Thanks to AFOSR & DTRA et al for Funding

34. www.youtube.com Deinococcus radiodurans Daly Deinococcus: A direct challenge to evolutionary theory "Creation Moments" daily 2 minute radio/broadcast with host Ian Taylor is heard around the world on over 1300 stations and outlets. Each program features scientific evidences of nature that point to delicate design not evolutionary chance. Creation Moments http://www.creationmoments.net/ “Another wonder of God's design that will not make it into middle or high school textbooks. Like a lot of other facts, Deinococcus just doesn't fit with the Religion of Evolutionism..”

35. Research Slide # 5 Bacillus Spores Contain Enzyme-Protecting Mn & Ca Complexes (DPA) kGy Mn2+ kGy Dipicolinic acid Putative Structures: 20% Ca2+ kGy Mn2+ Ca2+ For comparison, Blue structure is uridine

36. The Really Big Question: Death by Protein Damage in Mammalian Cells? For many oxidative stress conditions, DNA is no longer considered the principal target of ROS (radiation, bleach, H2O2, Fe2+, Cu2+, etc) in prokaryotes that accounts for their toxicity. These trends parallel some of those beginning to emerge for mammalian cells. For example, ● The relationship between DNA damage and -ray dose in human cells is about the same as in all other cell-types (0.005 DSBs/Gy/Genome). ● In cultured mouse cells exposed to -rays, protein oxidation precedes DNA damage, and is implicated as a critical and very early event in radiotoxicity. The new paradigm of radiation toxicity may apply to humans: The key to surviving radiation: Protect your Proteins! And consider using Deinococcus Mn complexes!

37. M. J. Daly and K. W. Minton (1995) Resistance to radiation. Science 270, 1318

38. M. J. Daly (2010) Revising the molecular basis for radiation effects on cells. Horikoshi, K. (Ed.). Springer, Japan: “On November 8, 1895 Wilhelm C. Roentgen was studying the passage of an electric current through a vacuum tube at the Physical Institute of the University of Wurzburg, Germany. He noticed that, if the Crookes tube was wrapped in black cardboard in a dark room, a barium platinocyanide screen located a few feet away glowed softly. Because the nature of the invisible light emanating from the Crookes tube was then unknown, Roentgen gave them the name X-rays. Within weeks, his discovery was an international news story; within months, Roentgen’s original experiment was being treated as a novelty. Thomas A. Edison arranged a special exhibit on Roentgen rays at the annual Electrical Exhibition in New York City’s Grand Central Palace in May 1896. This exhibit was a public sensation, mainly due to his demonstrating on a fluorescent screen the shadows of the bones of the hands of visitors. The early success and acceptance in the practical use of the X-ray in medicine was facilitated by such public displays. Unfortunately, the dangers of X-rays were not recognized until too late”. Wilhelm Röntgen(1845–1923) 1901 Nobel Prize in Physics

39. Glass beaker + 15,000 Gy Control D. radiodurans survives 15,000 Gy