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

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

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
Catastrophic
Fukushima
Nuclear
Accident
Level 7
April 8, 2013:
Another major radioactive tank leak discovered
Leaking Cold War Radioactive Waste
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

Extreme Ionizing Radiation Resistance
Since 1956, 42 distinct species of Deinococcus reported
3 days, 25 o C, 60 Gy/hour on nutrient agar
137 Cs
:
Kingdom Bacteria
Phylum: Deinococcus-
Thermus
Order: Deinococcales
Genus: Deinococcus
Species: D. radiodurans
(Oregon, USA, 1956)
60 Gy/hour

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

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

The Model ROS Production under Aqueous Conditions by Ionizing Radiation (x-Rays & g
-Rays) complexes

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

100%
Representative
Insects
Rotifers
C. elegans
Fungi
Halobacteria
Cyanobacteria
Deinococci
Amoebae
You and most of Life
72 DSBs <1 DSB 5 DSBs 400 DSBs 120 DSBs g
-rays
10% g
-Rays
UVC
Desiccation
0 1 10 100 1000 10000 Dose/Gy
0 0.1 1 10 100 1000 Dose/J/m 2
0 0.1 1 10 100 1000 Dose/days

So, the central question became: What is the molecular basis of extremely efficient DSB repair in Deinococcus ?
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.

g
~0.004 DSB/Gy/Haploid Genome
D. radiodurans
17,500 Gy
20 kb
D. radiodurans
Post-17,500 Gy
24 hours later

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 Mn 2+ (
0.25-1 mM
) in cytoplasm, but Fe out.
● As resistance in different bacteria went up, so did their [Mn ]
Mn 2+
Fe

Yet, for a given dose of g
-radiation, the level of protein damage in Deinococcus compared to other bacteria is very different
Resistance
Fe
Mn
The Bacteria
Exposed to
Same Dose
Same Protein
Purification
Procedure
Protein
Oxidation
Assay
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.

The Model
Protein Damage complexes

Something in D. radiodurans protein-free ultrafiltrates protects proteins
In vitro Mn 2+
+
!
+
!
!
+

Approach to Isolating Protective Mn 2+ Complexes in Extremely Radiation Resistant Organisms
Protein-Free Extracts Small-Molecule Analysis
Ultrafiltrates
Ultracentrifugation
+ Ultrafiltration
HPLC
MS - Metabolomics
Chromatography
Atomic Abs Spec
What is enriched?
Mn(II)
Orthophosphate
Peptides
Nucleosides
Applications
Radioprotection
Enzyme storage
Vaccine preparation
Reconstituted Complexes
Enzyme radiation activity assays. Carbonyl assays.
Radioprotection of human cells and bacteria.
In vitro and in vivo screening

Composition of the DR-ultrafiltrate d
Mn complexes
Free amino acids and peptide-derived amino acids

2+
Orthophosphate
Nucleosideanalogs O
2
· -
O
2
· HO·
Amino acids/
Peptides

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, Mn 2+ :
● is bound to orthophosphate not polyphosphate
● is bound to water (+)
● mainly bound to small molecules (SM) not enzymes
● coordinated with N (but probably also C=O)
● coordination is not altered by megadoses of g
-rays
E.coli S.cerevisiae
(Sensitive) (Moderately resistant)
+++
+++
+++
++ enzymes
- - -
SM
- - altered!
altered a bit

+
Then,
Bam HI
+ l
( Bam HI Assay)
For example:
25 mM PiB
25 mM
+ Bam HI
3 mM U
+ Bam HI
3 mM U
1mM MnCl
2
25 mM PiB
1mM MnCl
2
25 mM
25 mM PiB
3 mM U
1mM MnCl
2
+ Bam HI + Bam HI + Bam HI
Mg 2+ , Ca 2+ , Zn 2+ , Ni 2+ , Cu 2+ and Fe 2+ have no protective effect when combined with uridine (U) and PiB (phosphate buffer, pH 7.4)

Glutamine Synthetase
Aqueous Glutamine Synthetase (GS) Mixture:
25 mM PPB (pH7.4):
25 mM PPB (pH7.4)
25 mM PPB (pH7.4)
25 mM PPB (pH7.4)
25 mM PPB (pH7.4)
+ 1 mM Mn :
+ 1 mM Mn + 10 mM Leu :
+ 1 mM Mn + 3 mM (A+U):
+ 1 mM Mn + 3 mM Decapeptide:
New Designer
“Deinococcus
Peptides (NIH) + Mn +Pi
!!!
D
20
GS activity/ g
-ray dose:
150 Gy
1,800 Gy
>15,000 Gy
>25,000 Gy
>40,000 Gy
>100,000 Gy!!

100%
Representative
Insects
Rotifers
C. elegans
Fungi
Halobacteria
Cyanobacteria
Deinococci
Amoebae
You and most of Life
72 DSBs <1 DSB 5 DSBs 400 DSBs 120 DSBs g
-rays
10%
UVC
Desiccation
0 1 10 100 1000 10000 Dose/Gy
0 0.1 1 10 100 1000 Dose/J/m 2
0 0.1 1 10 100 1000 Dose/days

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

Application

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

MRSA MRSA

VEE VIRUSES

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

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.
No Radiation
E. coli
+ 42 Gy/hour
D. radiodurans
+ 42 Gy/hour
D. radiodurans
E. coli
+ 42 Gy/hour

● 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.

Latest information: : Daly + Deinococcus
Daly et al ., Science 306 , 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

www.youtube.com
Deinococcus radiodurans
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..”

Research Slide # 5
Bacillus Spores Contain Enzyme-Protecting Mn & Ca Complexes
(DPA) kGy
Mn 2+ kGy
Dipicolinic acid
20%
Putative Structures:
Ca 2+ kGy
Mn 2+ Ca 2+
For comparison,
Blue structure is uridine

For many oxidative stress conditions, DNA is no longer considered the principal target of ROS (radiation, bleach, H2O2, Fe 2+ , Cu 2+ , 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 g
-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 g
-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!

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

Wilhelm Röntgen (1845 –1923)
1901 Nobel Prize in Physics
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 Xrays were not recognized until too late ”.

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