Scientists from Arsenic Bacteria Paper Respond to Criticisms

Article Updated: 20 Jan , 2016
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Backlash from the “arsenic life” paper that was published on December 2, is still ongoing. Some of the criticism has been about the science, while much more criticism has been about the coverage of the news and also how NASA introduced, or “teased” the public with news, using the words “astrobiology” and “extraterrestrial life” in their announcement of an upcoming press conference. Today, at the American Geophysical Union conference, one of the team scientists, Ron Oremland discussed the fallout from the news coverage, and I’ll be providing an overview of that shortly. At about the same time, the science team released a statement and some FAQ’s about the science paper. Below is that statement and the information the science team provided.

Response to Questions Concerning the Science Article, “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorous”

-As of December 16, 2010-

A research article published December 2, 2010 by the journal Science provided several lines of evidence, collectively suggesting that a bacterium isolated from California’s Mono Lake can substitute arsenic for a small percentage of its phosphorus and sustain its growth.

This finding was surprising because six elements—carbon, oxygen, hydrogen, nitrogen, sulfur and phosphorus—make up most of the organic molecules in living matter, including nucleic acids, proteins and lipids. Scientists not affiliated with the research team have therefore asked appropriately challenging questions about the research.

A key purpose of scholarly publication is to advance science by presenting interesting data and proposing testable hypotheses. Understandably, the most surprising findings tend to generate the most intense response and scrutiny from the scientific community. Post-publication responses to original research, and efforts to test and replicate the results, especially in cases of unexpected findings, are an essential mechanism for advancing scientific knowledge.

Science editors have now received a number of technical comments and letters responding to the article, “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus,” by Felisa Wolfe-Simon and colleagues. The comments and responses will undergo review, and we will publish them in a future issue of Science.

Meanwhile, in an effort to promote public understanding of the work, the research article and a related news piece have been made freely available to the public via the Science Web site for the next month. These articles can be found online here:

Research article

News article

The Wolfe-Simon team, theorizing that perhaps some bacteria might be able to use arsenic or tolerate some substitution for phosphorus in organic molecules, collected microbes from arsenic-rich Mono Lake and then gradually weaned them off phosphorus, feeding them arsenic instead. The team has reported that they took steps to rule out any phosphorus contamination. They concluded that their evidence suggested arsenic had replaced a small percentage of the phosphorus in their DNA.

Various types of evidence were described by the authors, including:

* Inductively coupled plasma mass spectrometry.

The authors reported that these results revealed arsenic was inside bacterial cells, suggesting it was not merely a contaminant stuck to the exterior of the cells;

* Radioactive labeling of arsenic.

Wolfe-Simon’s team said this evidence allowed them to spot the normally toxic substance within the protein, lipid, nucleic acid and metabolite fractions of the cells, suggesting it had been taken into molecules forming each fraction.

* High-resolution secondary ion mass spectrometry of the DNA after it had been separated from the bacteria.

The authors reported that this evidence suggested the isolated DNA still contained arsenic.

* High-intensity (synchrotron) X-ray analysis.

Based on this evidence, the authors concluded that arsenic in the bacteria did appear to be replacing phosphates in DNA and other molecules.

Questions about the findings have tended to focus on whether the bacteria had truly incorporated arsenic into the DNA and whether the microbes had completely stopped consuming phosphorus. While the team prefers to address questions through a peer-reviewed process, Felisa Wolfe-Simon and Ron Oremland have provided some additional information here as a public service, and to clarify their data and procedures. Science emphasizes that these responses have not been peer-reviewed; they are provided on behalf of the authors only as a public information service while more formal review of their responses to comments sent to Science continues.

Preliminary Q&As

Question: Some people have questioned whether the DNA was sufficiently cleaned by your technique using gel electrophoresis, to separate it from other molecules. Do you feel this is a valid concern?

Answer:

Our DNA extraction and purification protocol begins with washed cells, pelleted from media. These are then subjected to a standard DNA extraction protocol, which included multiple phenol chloroform steps to remove impurities, including any unincorporated arsenate (As). After this, the DNA was electrophoresed, further separating the DNA from impurities. Any residual As from the media would have been removed by washing the cells prior to extraction and by partitioning into the aqueous phase during the 3 phenol:chloroform steps in the extraction. If As was incorporated into a lipid or protein it would have partitioned into the phenol, phenol:chloroform, or chloroform fractions. Additionally, DNA extracted in this manner on other samples was also successfully used in further analyses, including PCR, that require highly purified DNA.

The arsenic measured by NanoSIMS in the gel band is consistent with our other measurements and another line of evidence.

Our radiolabeled 73AsO43- experiment showed that of the total radiolabel associated with the cell pellet 11.0 % ± 0.1 % was associated with the DNA/RNA fraction. This indicated that we should expect some arsenate of the total pool associated with the nucleic acids. To interpret these data, we coupled our interpretation with our EXAFS evidence suggesting that intracellular arsenic was As(V) bound to C, and was not free in solution as an ion. This suggests the As is in, an organic molecule with bond distances consistent with a chemical environment analogous to phosphate (Figs. 3A, S3 “bond lengths” table). Further supporting our interpretation of the previous mentioned two analyses, we used a third line of evidence from NanoSIMS, a completely different technique from the other two. We find elemental arsenic (as measured by NanoSIMS) associated with the gel band that is more than two times the background in the gel. Based on the above discussion, we do not feel this is a valid concern.

Question: Others have argued that arsenate-linked DNA should have quickly fallen apart when exposed to water. Could you address this?

Answer:

We are not aware of any studies that address arsenate bound in long chain polyesters or nucleotide di- or tri-esters of arsenate, which would be directly relevant to our study. Published studies have shown that simple arsenic esters have much higher hydrolysis rates than phosphate esters (1-3). The experiments published to date have specifically looked at the exchange or hydrolysis of alkyl tri-esters of arsenate [Eqn. 1] and alkyl di-esters of arsenite [Eqn. 2]:

OAs(OR)3 + H2O ? OAs(OH)(OR)2+ ROH [1]

OAs(OH)(OR)2 + H2O ? OAs(OH)2(OR) + ROH [2]

where R = methyl, ethyl, n-pentyl and isopropyl. Reference 2 demonstrated that the hydrolysis rates for these simple alkyl triesters of arsenate decreased with increasing carbon chain length (complexity) of the alkyl substituent (methyl > ethyl > n-pentyl > isopropyl). No work has been done on the hydrolysis rates of arsenate-linked nucleotides or other biologically relevant moieties.

If the hydrolytic rate trend reported in Ref. 2 continues to larger-weight organics, such as those found in biomolecules, it is conceivable that arsenate-linked biopolymers might be more resistant to hydrolysis than previously thought. The small model compounds investigated in Refs. 1-3 are relatively flexible and can easily adopt the ideal geometry for water to attack the arseno-ester bond. Arsenate esters of large, bio-molecules, however, are likely to be more sterically hindered leading to slower rates of hydrolysis.

This type of steric constraint on reaction rate accounts for the wide range of rates seen in the behavior of some phosphate linked nucleotides. In small ribozymes, the phophodiester linkages at the site of catalysis can be hydrolyzed on the order of tens of seconds (with a chemical rate of 1 s-1). This rate enhancement is achieved by orienting the linkage for in-line attack by a nucleophile (an adjacent 2′ hydroxyl group). Moreover, the autodegradation patterns are consistent with specific base composition. On the other hand, the hydrolysis rates for phosphodiester bonds in A form duplexes of RNA are many orders of magnitude slower, because these linkages cannot access easily the geometry necessary for hydrolysis.

The rates in DNA may be much slower than model compounds because of the geometrical constraints imposed upon the backbone by the helix.

The kinetics of the hydrolysis of arsenate-linked biopolymers is clearly an area where more research is warranted.

Question: Is it possible that salts in your growth media could have provided enough trace phosphorus to sustain the bacteria?

Answer:

The data and sample labeling in Table S1 has caused some confusion. To clarify, for every experiment, a single batch of artificial Mono Lake water was made with the following formulation: AML60 salts, no P, no As, no glucose, no vitamins. Table S1 shows examples of ICPMS measurements of elemental phosphorus (~3 µM) and arsenate made on this formulation prior to any further additions. Then we added glucose and vitamins for all three treatments and either As for the +As treatments or P for the +P treatments. The P measurements made on the medium after the addition of sucrose and vitamins and after addition of As were also ~3 µM in this batch. Therefore, it was clear that any P impurity that was measured (~3 µM, this was the high range) came in with the major salts, and that all experiments contain identical P background (including any P brought in with the culture inocula).

In the Science paper, we show data from one experiment of many replicated experiments that demonstrates no growth of cells in media without added arsenate or phosphate (Figure 1). These data clearly demonstrate that strain GFAJ-1 was unable to utilize the 3µM P to support further growth in the absence of arsenate. Moreover, the intracellular P content determined for the +As/-P grown cells was not enough to support the full requirement of P for cellular function.

Note on culturing: All experiments were initiated with inocula from sustained +As/-P conditions. Prior to the experiments, the cells had been grown long term, for multiple generations from a single colony grown on solid media with no added phosphate. Before this, they were grown as an enrichment for more than 10 transfers and always into new medium that was +As/-P. We therefore feel that there is not significant carry-over of P. We also argue that there would not have been enough cellular P to support additional growth based on an internal recycling pool of P.

Question: Is there anything else you’d like for the public to understand about your research, or about the scientific process?

Answer: For all of us, our entire team, what this was like was unimaginable. We are a group of scientists that came together to tackle a really interesting problem. We each used our talents, from technical prowess to intellectual discussion, to objectively determine what exactly was happening in our experiments. We freely admitted in the paper and in the press that there was much, much more work to do by us and a whole host of other scientists. The press conference even included a technical expert, Dr. Steven Benner, who voiced some of the concerns we responded to above. Part of our reason for bringing this work to the community was to make the intellectual and technical connections for more collaborations to answer many of the lingering questions. We were transparent with our data and showed every datum and interesting result. Our paper’s conclusions are based on what we felt was the most parsimonious way to interpret a series of experiments where no single experiment would be able to answer the big question. “Could a microbe use arsenic in place of phosphorus to sustain its growth?” The best science opens up new questions for us as a community and sparks the interest and imagination of the general public. As communicators and representative of science, we feel that support of new ideas with data is critical but also to generate new ideas for others to think about and bring their talents to bear on.

We look forward to working with other scientists, either directly or by making the cells freely available and providing DNA samples to appropriate experts for their analyses, in an effort to provide more insight into this intriguing finding.

References

1. T. G. Richmond, J. R. Johnson, J. O. Edwards, P. H. Rieger, Aust. J. Chem. 30, 1187 (1977).

2. C. D. Baer, J. O. Edwards, P. H. Rieger, Inorg. Chem. 20, 905 (1981).

3. J.-M. Crafts, Bull. Soc. Chim. Fr. 14, 99 (1870).

4. R. Lagunas, D. Pestana, J. C. Diez-Masa, Biochemistry 23, 955 (1984).

Source: Felisa Wolf-Simon’s website, Iron Lisa

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12 Responses

  1. Olaf says:

    This is science at its best. A paper is produced, peer review can now check and repeat the experiments and if it cannot be reproduced by many different teams then it gets thrown in the garbage can.

    Pseudo scientists, would avoid publishing it for peer review, would create a web site an write a book how evil scientists are against them to hide the truth and that all science is wrong.

  2. Split_Infinity says:

    NASA and the scientists in it want funding, an appealing way to do that is to plant the idea of the possibility of life is just around the corner no matter how remote the chances actually are, this way they get public support that funding.

    • Torbjorn Larsson OM says:

      Conspiracy theories looks like a conspiracy! (O.o)

      Of course they want funding, that doesn’t implicate their specific claims. That there are other biospheres is not a far fetched idea, and that we can find out some of the possibilities by studying available life isn’t either. (But arsenic stuff … likely not. For one, As is ~ 1/1000 P in the universe.)

  3. Torbjorn Larsson OM says:

    Good for them. Unfortunately I can’t see that the provided information answers the raised questions, in some cases they are aware of that. Considering Redfield’s critique:

    Sufficient clean: They can’t or won’t explain why RNA is still prevalent in the washed samples as in the As-/P+ gel, alternatively don’t show up in the As+/P- gel. AFAIU the RNA should go out the way they propose the As go (aqueous phase).

    Arsenic-DNA stability. No evidence for or against (and they admit), but if non-DNA compounds falls apart the simple prediction is that so does DNA. Found As is consistent with impurities (see above).

    P pool. No evidence against, there are bacteria (including GFAJ, I think) that would survive on the available P.

    And what does this mean:

    For all of us, our entire team, what this was like was unimaginable.

    If it refers to the discovery, it is exactly what they imagined. If it refers to the reactions, it is exactly what they should have expected/wanted/accepts.

    Oh well, after the initial hiccups I’m sure it will be trashed out. At least they narrow their claim to something all can agree on, their data _may_ support some arsenic substitution (but probably not).

  4. Hon. Salacious B. Crumb says:

    As I said before.

    “IMO, I think the kind of story just feeds the non-scientifically trained sceptics, and just adds more confusion that science and scientific announcements are untrustworthy or are ‘faith-based.’ The continued misleading damage this does in the minds of the general public is immeasurable — to the extent they don’t trust science, the technology it produces, and how the world works. It makes all the complex work done in universities, laboratories and science institutions all that so much harder. In the end, if when true alien life is found, it is somewhat tarnished by those clambering for the spotlight just to make their research more appealing for the sake of ego.”

    …and now the truth of this story comes out. There is NO link here to alien life — real or otherwise! There is no grand new scientific breakthrough. It is all hype! All we have left is real doubt, and the unknowing public via the media, since 1st December is now out there believing a “newly discovered truth” that is just means absolutely dididly-squat!

    As to unbelievably saying; “The best science opens up new questions for us as a community and sparks the interest and imagination of the general public.”

    What Freudian slip!! Surely the “best science” is to design an experiment that can lead to some definitive conclusion, that can then be tested and reexamined to confirm it is true from the evidence available. Only then can you raise these “new questions” and start down some new avenue of research.

    What the hell has this to do with “…sparks the interest and imagination of the general public.” That isn’t on the scientific method manual, is it? All it means folks, is you want the public to know about your work so your funding is continued. You’ll do anything, say anything, to makes sure the money keeps rolling in so you don’t lose your job, your research grant, or your need for personal kudos. This is not ‘research’, it is actually blood-sucking marketing! 🙁

    Frankly, it has little to do with “best science” (which, as we have shown, is already dubious “opinion” anyway.)

    In the end I really feel like I’ve been duped, wasting my time on some dead-end avenue that has nothing to do with astrobiology or proving alien life. From the weak responses made here (in the scientists words) your just treating me as if I’m stupid!

    Surely the final media release was properly scrutinised by the scientists who were doing the experimentation? (If you didn’t, then YOUR the ones at fault for making sure your work wasn’t being misrepresented?) Blaming NASA who presumably; “… introduced, or “teased” the public with news, using the words “astrobiology” and “extraterrestrial life” in their announcement of an upcoming press conference.” just looks like avoid responsibility for what was “discovered.”

    If the NASA organisation was misrepresenting the experiment then you should have said something earlier!

  5. Dave says:

    @Salacious the authors never made those accusations at NASA those words were written by Nancy Atkinson in an article about the story, but blaming NASA was never attributed to the authors as you claim. Get your facts straight next time before you go on a righteous tirade.

    here is the article it appeared other places too I think: http://www.universetoday.com/81783/scientists-from-arsenic-bacteria-paper-respond-to-criticisms/

    • Hon. Salacious B. Crumb says:

      Dave,
      I think you have misread me here. The article we refer to is the MediaAdvisory M10-167 by NASA BEFORE this article was released. I.e. http://www.nasa.gov/home/hqnews/2010/nov/HQ_M10-167_Astrobiology.html

      Here the words clearly state;

      “WASHINGTON — NASA will hold a news conference at 2 p.m. EST on Thursday, Dec. 2, to discuss an astrobiology finding that will impact the search for evidence of extraterrestrial life. Astrobiology is the study of the origin, evolution, distribution and future of life in the universe.”

      The argument is exactly as I and others have said; this story and paper has little to do with “astrobiology” nor “extraterrestrial life.”

      The problem here lies with the NASA media people who released this. (The paper in question doesn’t even mention “astrobiology” nor “extraterrestrial life.” My point was; How much did the the authors know about the allegedly wrongly worded NASA’s pre-release announcement?
      By them saying; “The best science opens up new questions for us as a community and sparks the interest and imagination of the general public.“; suspiciously infers that they did, and so my statement; “…then you [authors] should have said something earlier!

      So please. Where is the evidence to the contrary, here?

      • Billy says:

        Well, their discovery does exactly what NASA said:
        finding that will impact the search for evidence of extraterrestrial life

        it impacts the search because now, arsenic-rich worlds -could- harbor life, where as before we wouldn’t have looked there. Or, finding a large concentration of arsenic that would not have occured naturally.

        They only said that it impacts the search and/or impacts the search of evidence. 🙂

      • Hon. Salacious B. Crumb says:

        Well, their discovery does exactly what NASA …
        I disagree. If the science is doubt, so you can’t say that.
        The only impact it has is that science needs, is to be far more careful in claiming new discoveries before going to press.
        If you swallow and believe this story is true, then I think you have been conned into the propaganda machine instead of the facts as they are presented.
        IMO, just looking at the body language from the Felisa Wolfe-Simon tells me something within their paper is dubious or something else is not quite right with this story. She looked far too uncomfortable, immediately suggesting avoidance or deception.
        So let’s wait until the repeating of the experiment is done. (Considering the number of steps to reach their conclusion, n unforeseen or unknown slip-up does seems quite possible)
        I for one will remain quite sceptical of this story.
        As for your own speculations of its importance, well, I just hope you are right!

  6. Dave says:

    Salacious, in your post you certainly seem to imply that the authors are pointing fingers at NASA but in reality that never happened.

    “Blaming NASA who presumably; “… introduced, or “teased” the public with news, using the words “astrobiology” and “extraterrestrial life” in their announcement of an upcoming press conference.” just looks like avoid responsibility for what was “discovered.” ”

    You are correct that the original paper never mentions astrobiology, however I would point out that the term “astrobiology” in NASA-type researcher circles probably means something different than it does to the general public. As you quote in your last post, the field of astrobiology studies possible forms of life that may someday be discovered in the universe. There is even a journal called “Astrobiology” that publishes peer-reviewed science concerning extremophilic bacteria and other topics relevant to the search for potential life. So, when NASA was saying that this study was relevant to astrobiology I believe that they were trying to use the term in this sense (personally I don’t like this term since no “astrobiological” life has yet been discovered, but it is an established term for this type of research). Should they have realized that it would be misunderstood by the lay press? Yes, probably, but I don’t think they were trying to deceive anyone.

    • Hon. Salacious B. Crumb says:

      Dave
      Actually, thinking about it for a while.. No I am not pointing fingers at NASA, I am pointing fingers at the NASA press release. It is the process of “dumbing down” that is the issue — and the incorrect use of the words; “astrobiology” and “extraterrestrial life.”
      These were added just to catch the wider media attention so the story would “go viral.” It successfully did.
      Sadly the dichotomy between the actual scientific paper and the press release is so large, that the claims can almost be categorically be debunked. I.e. The science was not (as yet) validated nor proven beyond all doubt.
      In science “deception” has a different meaning. It does not refer only to the “conclusion.” It also refers to the process to make that “conclusion.” I.e. If one takes a deliberate short-cut during the experimentation or ignores, say, some “bad data.”
      Perhaps the word “deception” is wrong. Perhaps is is more correct to say “stretching the whole truth” a bit!

      As for; “Should they have realized that it would be misunderstood by the lay press? Yes, probably, but I don’t think they were trying to deceive anyone.

      Well yes and no. You cannot deceive anyone if you don’t have all the facts in front of you. The press just hopes what they are given is the absolute truth, when in fact, the press and pre-press release were already dumbed down for novice consumption. Again, especially adding “extraterrestrial life” to the story, shows intent of attention grabbling. (Also I haven’t use the strong word “deceive” per se, in all my responses here.)

      Regardless, I do appreciate you expressing point of view here, and agree somewhat with the sentiments of the often difficult issues of dealing with language semantics. You make some good points.

  7. Lawrence B. Crowell says:

    My main criticism of this article is that I don’t think they really demonstrated that arsenate was incorporated into DNA and in the molecular pathways, such as kinase phosphorylation. This may indeed be the case, but further experimentation is needed to conclude that.

    LC

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