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

Deinococcus Mn2+ Complexes: New Frontiers

Michael J. Daly

Uniformed Services University of the Health Sciences (USUHS)Bethesda, MD 20814, USA

Email: michael.daly@usuhs.edu

Deinococcus Mn2+ Complexes: New Frontiers

Michael J. Daly

Uniformed Services University of the Health Sciences (USUHS)Bethesda, MD 20814, USA

Email: michael.daly@usuhs.edu

Deinococcus History The Paradox

Mn2+ Antioxidants

Protein ProtectionApplications

MCB SeminarApril 25, 20133:30 pm - 4:30 pmLecture Room CUSUHS

Radiation-ResistantDeinococcusradiodurans

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

Leaking Cold War Radioactive Waste

Nuclear Threats

Catastrophic Fukushima Nuclear Accident

Burns from radiation devices known since Thomas Edison’s public displays of x-rays in 1896

Level 7

March 2011: Reactors # 1, 3, 4

April 8, 2013:Another major radioactivetank leak discovered March 10, 2013:

Major radioactivetank leak discovered

March 26, 2013:North Korea aims nuclear missile at US

Extreme Ionizing Radiation ResistanceSince 1956, 42 distinct species of Deinococcus reported

Kingdom:

Bacteria

Phylum: Deinococcus-Thermus

Order: Deinococcales

Genus: Deinococcus

Species: D. radiodurans(Oregon, USA, 1956)

3 days, 25oC, 60 Gy/hour on nutrient agar

Not

ting

ham

Ant

arct

ica

D. g

rand

isD

. pro

teol

ytic

usD

. radiopugnans

Port

ugal

D. roseus

D. r

adio

philu

sD

. geothermalis

137Cs 60 Gy/hourJapanPeru

N. A

tlantic

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

0.5 m

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

Hab

er-Weiss

Reaction complexes

The Model ROS Production under Aqueous Conditions

by Ionizing Radiation (x-Rays & -Rays)

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

0.0005 DSB/Gy/Mbp

0.05 DSB/Gy/Mbp

0.2 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

DSB Yields for -Rays

Hu

man

lun

g fib

rob

lasts0 1 10 100 1000 10000

100%

10%S

hewanella oneidensis

Escherichia coli

Dein

oco

ccus rad

iod

uran

s

Sur

viva

l

Dose/Gy

Caenorhabditis elegans

Survival Curves and DSBs

Representative Insects Rotifers C. elegans Fungi Halobacteria Cyanobacteria Deinococci Amoebae

You and most of Life

0 0.1 1 10 100 1000 Dose/J/m2

-Rays

UVC0 0.1 1 10 100 1000 Dose/daysDesiccation

120 DSBs400 DSBs5 DSBs<1 DSB72 DSBs -rays

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.

nucleoidsgenes

Mn

Pigments

Mars

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

For a given dose of -radiation, the level of DNA damage in D. radiodurans compared to all other organisms is very similar

~0.004 DSB/Gy/Haploid Genome

20 kbD. radiodurans17,500 Gy

D. radioduransPost-17,500 Gy24 hours later

The

Par

adox

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

Yet, for a given dose of -radiation, the level of protein damagein Deinococcus compared to other bacteria is very different

Pro

tein

Ca

rbon

yl

The

Par

adox

A founding concept of radiobiology that deals with X-rays + -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 Bacteria

Exposed to Same Dose

Same ProteinPurificationProcedure

Resistance

Protein Oxidation Assay

Fe

Mn

Protein Damage

Hab

er-Weiss

Reaction complexes

The Model

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

Something in D. radiodurans protein-free ultrafiltrates protects proteins

-ra

diat

ion

-ra

diat

ion

desi

ccat

ion

Bam

HI

Bam

HI

EC

pro

tein

s +

Ultr

aftr

. +

+

+

In vitro Mn2+

Approach to Isolating Protective Mn2+ Complexes in Extremely Radiation Resistant Organisms

Ultrafiltrates

Ultracentrifugation + Ultrafiltration

HPLCMS - MetabolomicsChromatographyAtomic Abs Spec

Protein-Free Extracts Small-Molecule Analysis

In vitro and in vivo screening

Enzyme radiation activity assays. Carbonyl assays. Radioprotection of human cells and bacteria.

Radioprotection Enzyme storageVaccine preparation

Applications

Reconstituted Complexes

What is enriched?

Mn(II)OrthophosphatePeptidesNucleosides

Composition of the DR-ultrafiltrate

d Free amino acids and peptide-derived amino acids

Mn complexes

HO· O2·-

O2·-

Putative D. radiodurans Mn2+ Complexes

Amino acids/Peptides

Orthophosphate

Nucleoside-analogs

Using various forms of paramagnetic spectroscopy:

Professor Brian HoffmanDepartment of ChemistryNorthwestern University2145 Sheridan RoadEvanston, IL 60208-3113

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 enzymes SM

● coordinated with N (but probably also C=O) - - - - - - ● coordination is not altered by megadoses of -rays altered! altered a bit

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

25 mM

25 mM

Extraordinary Antioxidant Synergism (BamHI Assay)

For example:

Mg2+, Ca2+, Zn2+, Ni2+, Cu2+ and Fe2+ have no protective effect when combined

with uridine (U) and PiB (phosphate buffer, pH 7.4)

Then,

BamHI

+

3 mM U 1mM MnCl2

25 mM PiB

1mM MnCl2

3 mM U25 mM PiB 25 mM PiB

3 mM U1mM MnCl2

+ BamHI + BamHI + BamHI + BamHI + BamHI

+

Aqueous Glutamine Synthetase (GS) Mixture: D20 GS activity/ -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!!

Glutamine Synthetase

We were Stunned!

Hu

man

lun

g fib

rob

lasts0 1 10 100 1000 10000

100%

10%S

hewanella oneidensis

Escherichia coli

Dein

oco

ccus rad

iod

uran

s

Sur

viva

l

Dose/Gy

Caenorhabditis elegans

Reminder: Survival Curves and DSBs

Representative Insects Rotifers C. elegans Fungi Halobacteria Cyanobacteria Deinococci Amoebae

You and most of Life

0 0.1 1 10 100 1000 Dose/J/m2

-rays

UVC0 0.1 1 10 100 1000 Dose/daysDesiccation

120 DSBs400 DSBs5 DSBs<1 DSB72 DSBs

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

Application

Irradiated Vaccine (See next slide for NIH study)

Protein & Structure Survives!

Everything is wiped out

40,000 Gy 40,000 Gy+ Mn-pep-Pi

DNA Destroyed

+ DR Mn-Complexes (Mn-pep-Pi)

Immunogenic!

Lambda Phage

tailtail

DNA is destroyed

Proteins survive

Next Slide – Details of Collaboration between Daly and Datta Groups

All USUHS

MRSA MRSA

VEE VIRUSES

Cel

l Via

bilit

y %

1000%

Radiation Dose (Gy)0

Ex Vivo Radioprotective Effects of Reconstituted D. radiodurans Mn-Peptide Complexes on Human Cells100%

Collaboration between Daly and Tom Lamkin’s Group

DR-ultrafiltrate

Control

Reconstituted Complex

+ 42 Gy/hour

Mn-Complexes applied to the growth medium of E. coli endowsit 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

+ 42 Gy/hour+ 42 Gy/hour

E. coli

D. radiodurans E. coliD. radiodurans

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.

Daly et al., Science 306, 925-1084 (2004) Daly et al., PLoS Biology, 5(4) (2007) Daly et al., ISME J, 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)

Latest information: : Daly + Deinococcus

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..”

Daly

Research Slide # 5

Ca2+

Mn2+

Dipicolinic acid

(DPA)

Bacillus Spores Contain Enzyme-Protecting Mn & Ca Complexes

For comparison,Blue structure is uridine

20%

Mn2+ Ca2+

kGy

kGy

kGy

Putative Structures:

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!

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

“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

M. J. Daly (2010) Revising the molecular basis for radiation effects on cells. Horikoshi, K. (Ed.). Springer, Japan:

Glass beaker + 15,000 Gy Control

D. radiodurans survives 15,000 Gy