Kanai's Laboratory Literature Seminar

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Can Life use Arsenic
instead of Phosphorus ?
Kanai’s Lab. Literature Seminar
27th October 2012
Junya Kawai (M2)

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Index
1. Phosphorus in Life
2. NASA’s Report in 2010
3. Objections to NASA’s Report
4. Conclusion

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1. Phosphorus in Life

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Six Nutrients Elements of Life
Nitrogen2.6%*Calcium
1.4%

Phosphorus Sulfur
0.2%
1.1%

Others0.4%

Hydrogen10.0%
Carbon22.9%

Oxygen
61.4%Elements in human body (w/w %, IAEA, 1972)*Calcium is not included in “six nutrients”.

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5Phosphorus
Atomic number: 15
Atomic weight: 30.973762
Electron configuration: [Ne] 3s2 3p3
Atomic radius: 98 pm
Electronegativity (Pauling): 2.19
Stable isotope: 31P
Oxidation states: +V ~ -III (+V is most important for life.)
Allotropes: white, red, black, etc.
© periodictable.com

reagent© IUPAC (http://www.iupac.org/)

http://www.siyaku.com

fertilizer
http://www.onoda-kagaku.co.jp

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Phosphorus in LifeOONH2POOROPhosphateOPR'OO

Phosphodiester

NNH2NOOOONNONH

OOO PONH2NOOONOONO P OOHNOONH2OHOHO P ONOOOHOHNH2NNOOO P O

NNODNAOOATPNNONH2OO POOOO P O P O P O

NNNH

Proteins

O P O

NONO

NAD+O

OHOH

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Kanai's Laboratory Literature Seminar

Phosphodiester: an Anionic linker
O OPRR'OOOR

phosphodiesterNHamideOR'ROesterR'O OR'RSOOsulfonate

pKa ~2.1
cf .

O O HPHOOH

Phosphodiester can link two carbons
and still ionize.

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8Why DNA use Phosphorus
 Ionized phosphodiester is highly tolerant of hydrolysis.

- Rates of saponification at 35 oC

k (M-1s-1)
relative rate

O OPe
M OOMeO OMePeM OOMe2.0 x 10-9
1.0

3.4 x 10-4
2 x 105

OMeOEt1.0 x 10-25 x 106

 Phosphodiester is retained within membrane.

- Due to the lipophobicity of anion, molecules can be kept inside membrane.
 Molecules can be bound by electrostatic interaction- For example, the “packaging” of DNA around histone Phosphate is redox inactive in a physiological conditions.

- It can be reduced to phosphite only at potentials as low as -700 mV.
Westheimer, F. H. Science 1987, 235, 1173-1178.

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2. NASA’s Report in 2010

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NASA Astrobiology Institute (NAI)
https://astrobiology.nasa.gov/nai/ AstrobiologyThe study of the origin, evolution, distribution, and future of life in the universe
 Three questions they address- How does life begin and evolve?
- Is there life elsewhere in the universe?
- What is the future of life on Earth and beyond?
 What they do- The search for habitable environments in our Solar System and on planets
around other stars- The search for evidence of prebiotic chemistry or life on Solar Systembodies such as Mars, Jupiter’s moon Europa, and Saturn’s moon Titan- Research into the origin, early evolution, and diversity of life on EarthKanai's Laboratory Literature Seminar

11NASA’s Press Conference in 2010https://astrobiology.nasa.gov/nai/“NASA will hold a news conference at 2 p.m. EST onThursday, Dec. 2, to discuss an astrobiology finding thatwill impact the search for evidence of extraterrestrial life.”

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NASA’s Report on Science
Wolfe-Simon, F. et al.
Science 2011, 332, 1163-1166.

• They claimed that a bacterium GFAJ-1 could sustain its

growth by using arsenate instead of phosphate.
GFAJ-1
 A rod-shaped bacterium Isolated from the sediment of Mono lake.
 Belongs to the Halomonadaceae family,identified by 16S rRNA sequence philogeny.
Figure: SEM image of a strain of GFAJ-1
cultured under arsenic-rich condition

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Mono Lake© NASA• Located in eastern California, U.S. (http://www.monolake.org/)• An example of “extreme environments” on earth- Alkaline: pH 9.8- Hypersaline: 78 g/L (2.5 times higher than ocean)- Arsenic-rich: 200 µM (Highest in the world)• Some unnatural lives have already been found here.
As(III)-fueling photosynthesis: Oremland, R. S. et al. Science 2008, 321, 967-970.

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Growth of strain GFAJ-1-As/+P+As/-P• Cell growth was monitored
by both an increase in optical density
and cell numbers.
• Cell grew the fastest under –As/+P.

-As/-P-As/+P+As/-P-As/-P• Also under +As/-P, cell could grow to be
20-fold in cell numbers after 6 days,
while it didn’t grow under –As/-P.
 GFAJ-1 seems to use arsenate insteadunder phosphate-poor condition.
* Artificial medium (pH 9.8) contains 10 mM glucose,other vitamins and minerals required but no phosphate.
**+As: 40 mM AsO43+P: 1.5 mM PO43-P: 3.1 (±0.3) µM PO43- (background)
Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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Electron Microscopy of GFAJ-1

Vacuole-like regions were especially enlarged.

E: TEM** image of +As/-P
Cells became
1.5-fold larger.

** Transmission Electron Microscopy

C: SEM* image of +As/-P
D: SEM* image of -As/+P* Scanning Electron Microscopy***+As: 40 mM AsO43+P: 1.5 mM PO43-P: 3.1 (±0.3) µM PO43- (background)

Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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ICP-MS Analysis of Intracellular Elements
ICP-MS: Inductively Coupled Plasma Mass Spectrometry
Possible to detect the quantity of each containing element (or its isotope)(http://www.chem-agilent.com/contents.php?id=513)

They claimed followings:• Higher intracellular As & lower P were detected for the +As/-P cells.
• Intracellular P grown +As/-P is 96.5% less than that of –As/+P,
which is far below the required amount to support growth (1-3 % P by dry weight).
Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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Elemental Distribution• Radiolabeled 73AsO43- was employed to check the intracellular distribution.

DNA may contain 73As.

Proteins & small molecule weight metabolites(NADH, ATP, acetyl-CoA etc.) may be arsenylated.

Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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NanoSIMS Analysis of DNA• The extracted DNA/RNA fractions were loadedon agarose gel and the bands indicated wereanalyzed.
•75As ion ratio of +As/-P relative to 12C was twice

as much as that of –As/+P.
(The value of –As/+P is almost same as blank.)•

31P:12C ratio was 3.4 times less than –As/+P.

 Purified DNA from +As/-P seems to contain As.

Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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XANES & EXAFS Analysis
 XANES : X-ray Absorption Near Edge Structure
Electron status or symmetry can be detected.
• As(V) coordination was detected.
 EXAFS : Extended X-ray Absorption Fine Structure
Coordination number and atomic distance can be obtained.

• The first neighbor shell around As consisted of
4 oxygen ligands and had a second shell, whichis inconsistent with previous data.
• But authors said its spectrum matched withthat of the model structure of arsenylated
DNA (red), metabolites, or proteins.

FT magnitude(http://support.spring8.or.jp/Doc_lecture/PDF_090127/xafs_4.pdf)

Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

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Arsenic
Atomic number: 33
Atomic weight: 74.92160
Electron configuration: [Ar] 4s2 3d10 4p3
Atomic radius: 114 pm
Electronegativity (Pauling): 2.18
Stable isotope: 75As
Oxidation states: +V ~ -III
Allotropes: grey, yellow, black© periodictable.com© IUPAC (http://www.iupac.org/)

Salvarsan (drug)
Semiconductor (Gallium arsenide)© Science Museum, London http://www.theodoregray.com/

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Kanai's Laboratory Literature Seminar

Arsenate vs Phosphate Similarities• Same electron configuration (s2p3 for unclosed shell)• Nearly identical pKa (As: 2.2, 6.97, 11.53 / P: 2.1, 7.2, 12.7)• Thermochemical radii (only 4% different) Differences• Less tolerance for hydrolysis- Kinetic stability of arsenate in H2O
is far below that of phosphate.
Edwards, J. O. et al. Inorg. Chem. 1980, 20, 907.
Gates, K. S. et al. ACS Chem. Bio. 2011, 6, 127.

• Easily reduced to As(III)
RO
RAs
O OO

H2OROHOAsOOR

fastOH- ROHROOHAsO O

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Summary of NASA’s Claim• Cells grew under +As/-P, although they didn’t under -As/-P.
• Higher intracellular As & lower P were detected (than –As/-P).
• P value was less than required for its growth (1-3 % P by dry weight).
• Based on the purification by extraction, proteins, s.m.w. metabolites

and DNA might contain arsenic.
• NanoSIMS analysis of DNA also supported the fact above.
• XANES & EXAFS suggested the arsenate was incorporated in DNA

or other microorganisms.

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3. Objections toNASA’s Report23Kanai's Laboratory Literature Seminar

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Eight Technical Comments have come !!

All doubted whether GFAJ-1 was truly using arsenic in its DNA.

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1. Unsolved Matters
 Stability against hydrolysis of arsenate diester- Arsenic ester can be hydrolyzed in water much faster than phosphate.
- If the arsenylated DNA was stabilized by association with another molecule,
it would be required to remain associated with DNA through extraction andgel electrophoresis processes.
 High redox potential of As(V)- As(V) is reduced into As(III) in the physiological range of redox potential.
- The resulting structural change & oxidation of other molecules impair themetabolic processes.
 Relatively lower intracellular As concentration

Medium: As/P = ca. 10000
Cell: As/P = only <10Kanai's Laboratory Literature Seminar26

2. Inadequate Data & Analyzing Methods
 No direct evidence of incorporation of As in DNA
 Insufficient purification method of DNA- Aqueous DNA/RNA fraction they used directly is typically contaminated.
- It wasn’t purified from the agarose gel which may contain some elements.
 High phosphate concentration on background (ca. 3 µM)
 Unknown purity of arsenic
 Incorrect statistical calculation and large estimated errors

Error is larger than value itself !

Calculation of average is not correct, in fact !

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3. Alternative Interpretations
 Induced Pst system under +As/-P condition- Many bacteria have two systems to assimilate phosphorus:(i) phosphate inorganic transport (Pit): Low affinity for P and always active(ii) the phosphate-specific transport (Pst): High affinity for P and active whenphosphate levels are low- Arsenate is known to poison the Pit system and to accelerate Pst system.
Malamy, M. H. et al. J. Bacteriol. 1980, 144, 366.

- Growth under +As/-P is due to the increased uptake of P by Pst system ?
 Cell growth in volume perhaps means the storage of toxic molecules.
Nature 2010, 468, 741.

 Some bacteria only need much less than

1 % P by dry weight.

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Two Definitive Reports in 2012

Erb, T. J. et al. Science 2012, 337, 467-470.; Redfield, R. J. et al. Science 2012, 337, 470-473

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29“As-Resistant”, but still P-Dependent[µM PO43-]
15.4
1294 ~NASA’s“-P”
1.7<0.3with 40 mM
AsO43-

Black: +As (40 mM)
Red: -As<0.3• Growth correlated with the amount ofphosphate, and 1.7 µM is sufficient.
• +As/-P (<0.3 µM): No growth occurred.

• Final growth didn’t change whether
or not arsenate was added.
Erb, T. J. et al. Science 2012, 337, 467-470.

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30*+As: 40 mM+P: 10 µM-P: <0.3 µM(circle)(circle)“As-Resistant”, but still P-Dependent
P (medium)P(cell)+As/+P-As/+P+As/-P+As/+P-As/-P(diamond)(diamond)• Growth correlated with the amount ofphosphate, not with that of arsenate.

• During growth, phosphorus in themedium decreased and became
enriched in the cellular fraction.

 During growth, GFAJ-1 is obviously using phosphorus in the medium.
Erb, T. J. et al. Science 2012, 337, 467-470.

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Metabolomic Analysis by HRMS
• Cells were cultured in almost the same medium as reported.

(As: 40 mM(+) or 0 mM(-), P: 9 µM, glucose: 10 mM)• In both media, only phosphorylated metabolites were detected.

- Only nucleotide bisphosphates (ADP etc.) appeared elevated in thecell cultured +As, which might result from a higher energy demandwhen grown +As.
(Due to ATP-dependent detoxification or induced Pst system ?)• Although hexose arsenate was detected in MS, it was found this isabiotically formed glucose arsenate.

Erb, T. J. et al. Science 2012, 337, 467-470.

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Analysis of Well-Purified DNA• Independent of As and P, similar-sizedfragments are obtained in both double& single-stranded DNA .
• Hydrolysis or other stabilizing molecules
were not observed.
• According to the elemental analysis ofpurified DNA, no As was detected.

A.
B.
C.

Soon after purification
2 months later @ 4 oC
2 months @ 4 oC, 10 min. @ 95 oC
Redfield, R. J. et al. Science 2012, 337, 470-473

Erb, T. J. et al. Science 2012, 337, 467-470.

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As-induced Massive Ribosome Breakdown One reason why arsenate stimulated the growth of GFAJ-1 might bethat arsenate induces massive ribosome degradation, providing asource of phosphate.
*+P: 1.5 mM-P: 3.1 (±0.3) µM-As/+P+As/-P-As/-P

Long lag periodprior to start of growth
Wolfe-Simon, F. et al. Science 2011, 332, 1163-1166.

Two intensive bands disappearedin +As/-P. (23S and 16S rRNA ?)Deutscher, M. P. et al. J. Biol. Chem. 2012, 287, 28816.

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As-induced Massive Ribosome Breakdown
E. coli : known to be As-resistant to some extent
E. coli growing in 0.1 mM phosphate medium
was prelabeled with [3H]-uridine. (ribosome label)After washing, cultured under various media
Measured release of acid-soluble radioactive products.
Deutscher, M. P. et al. RNA 2011, 17, 338.

rRNA was degradated
to free base,providing Pi.

Samephenomena
as GFAJ-1+As/-P: After 80 h, small numbers of As-torelant cells
appeared and then started to increase.

Deutscher, M. P. et al. J. Biol. Chem. 2012, 287, 28816.

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Supposed Conclusion
A

B

A

B

A

B

A

B

C

DC

DC

DC

D-As>10 µM P+As>10 µM P

A

B

C

D

Pst
induced-As
3 µM P+As
3 µM P

A

B

A

B

C

D×

C

D

Pst
induced

B×C ×
D×
A

Pi istoo low.

A
C

A
C

B
D

B
D

A
A

B
B

C
C

A
C

A

B

Pi

D
A

growthgrowth

GFAJ-1

A

no growthslow growthRNAdegradation

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Summary of Objections• GFAJ-1 used contaminated phosphate in medium for growth.
• Higher intracellular As is due to the inadequate purification of DNA.
• Metabolomics & elemental analysis showed no incorporation of As

into DNA or other microorganisms.
• The reason arsenate stimulated the growth of GFAJ-1 might be either

massive ribosome breakdown or induced Pst system or both.
 GFAJ-1 is still phosphate-dependent.
• Although As was found to be inessential for that strain to grow,still we cannot deny the possibility that GFAJ-1 is using As for some purposes.

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Appendix: How Life differentiate P & As
• PBP (Phosphate Binding Proteins):- The important unit for recognition of phosphate in Pst system (more selective one)
- Discrimination of phosphate from arsenate is generally ca. 500-fold.
- GFAJ-1 has two types of PBPs, one of which has >4500-fold discrimination ability.

phosphate••
PBP of P. fluorescens (pH 8.5)
As (red & violet sphere), As-bound (green)
P (orange & red sphere), P-bound (pink)

Arsenate

The only difference is the direction of a high-energyhydrogen bond between O2-H & Asp62 anion.
Asp62 enables phosphate to bind more selectively
than arsenate.
Elias, M. et al. Nature, 2012 (doi:10.1038/nature11517)

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4. Conclusion

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Conclusion• The answer to the title question,“Can life use arsenic instead of phosphorus ?”--- is still unclear.

• At least, GFAJ-1 is not arsenic-dependent bacterium. It still needs asmall amount of phosphorus to grow.
• It is very important also for us

to select the proper methods to make the things confirmed,
to think whether alternative explanations for results are impossible,
and to conduct control experiments strictly.
• And, it seems that life system is much mightier than I thought.