In the wake of the NASA excitement over the new arsenic study, and my promise to give a detailed review of the paper itself, I have recruited a colleague with strong opinons about the work, a solid chemistry and microbiology background, and "Dr." in front of his name to share his analysis. I will be posting have posted my personal and less-technical take on the whole thing soon, so stay tuned as well.
Dr. Alex Bradley uses modern geochemistry and microbiology tools to study the evolution of life and Earth. He has the following to say about the paper.
There's been a lot of hype around the news of GFAJ-1, the microbe claimed to substitute arsenate for phosphate in its DNA. In the midst of all the excitement, one thing has been overlooked:
The claim is almost certainly wrong.
The study published in Science has a number of flaws. In particular, one subtle but critical piece of evidence has been overlooked, and it demonstrates that the DNA in question actually has a phosphate - not an arsenate -backbone.
To understand why, we need to back up a bit. One thing that everyone agrees on is that all things being equal, DNA with an arsenate backbone will hydrolyze quickly in water, while DNA with a phosphate backbone will not. Steve Benner has pointed out that the half-life of the hydrolysis reaction is about 10 minutes.
Wolfe-Simon et al. recognize this, but claim that the bacterium GFAJ-1 must have some unknown biological mechanism to compensate, and this prevents the DNA from falling apart in the cells. Let's assume for now that they are correct. It might be plausible - biology has all kinds of strange tricks and this idea can't be quickly dismissed, even if it seems radical.
But chemistry is much more predictable. Once DNA is out of the cell, pure chemical processes take over, and experiments have demonstrated that hydrolysis of arsenate links is fast. So you could do a simple experiment to test whether DNA had a phosphate or arsenate backbone: just remove DNA from the cell and put it in water for a few minutes. Then examine whether it hydrolyzes or not.
In an accidental way, Wolfe-Simon et al. performed precisely this experiment. The result indicates that the DNA of GFAJ-1 has a phosphate backbone.
The details are these: to isolate DNA, Wolfe-Simon et al. performed a phenol-chloroform extraction. In this technique, after cellular disruption, DNA and other cellular material were dissolved in water, and then the non-DNA material (such as lipids and proteins) were cleaned out of the mixture using phenol and chloroform. This is a pretty common laboratory procedure, and typically would take an hour or two. But here is the key point:
During this whole procedure, the DNA was in water.
Remember, proteins were removed from this mixture. Any cellular machinery that stabilized arsenate-DNA was removed. In the absence of biochemistry, pure chemistry takes over: any arsenate-DNA would have been quickly hydrolyzed in the water, breaking down into fragments of small size. Alternatively, phosphate-DNA would not hydrolyze quickly, and large-sized fragments might be recoverable.
So what size are the fragments of DNA extracted from GFAJ-1? They are large. Figure 1 shows a single strong band. This pattern is a bit unusual for a genomic DNA extract, but the important thing is that the fragments in this band have around 10,000 nucleotides between breaks in the DNA. These long chains of nucleotides did not hydrolyze in water. Yet it is precisely this DNA band that is claimed to have an arsenate backbone.
How can this be?
The answer is: it can't be. If this DNA did not hydrolyze in water during the long extraction process, then it doesn't have an arsenate backbone. It has a phosphate backbone. It is normal DNA.
So what accounts for the claim of arsenic in this DNA? Wolfe-Simon et al. used a technique called nanoSIMS to analyze elemental concentrations of the agarose gel at the location of the DNA band. They determined that the part of the gel containing DNA also contained both arsenic and phosphorus. But what did they really analyze?
The answer is that the nanoSIMS determined the concentration of arsenic in the gel - not specifically in the DNA. Arsenic was present in the gel at the location of the DNA band. But these data do not require that arsenic is part of the DNA, only that it is somehow associated with the DNA. So here is a more plausible explanation: arsenate sticks to stuff. When you grow bacteria in media containing lots of arsenate, cellular material gets covered in arsenate. If you analyze this material chemically, you see a high arsenic background. The arsenic background will remain even after you separate the cellular material into its constituent parts - DNA, lipids, and proteins - because the chemical separation is imperfect. You could imagine a parallel experiment: if you grew bacteria in seawater, a band of DNA extracted from these bacteria might show a high background of sodium and chloride. This would not be very surprising - and it certainly wouldn't imply that the DNA had a chloride backbone.
Wolfe-Simon and her colleagues might quibble with this, and claim that arsenate is not that 'sticky'. This should have been resolved by running a negative control. Grow some bacteria with phosphate-backboned DNA in media containing high concentrations of arsenate. Then extract the DNA, run a gel, and just demonstrate that the gel does not have a high arsenic concentration associated with the DNA band. That would be evidence that my explanation is wrong. But this simple control was not performed in study published in Science.
One objection to my claim might be: if the GFAJ-1 DNA contains phosphate, where did the phosphate come from? The researchers claim that there wasn't much phosphate in their growth media. In fact, they did a very good job of quantifying the background phosphate concentration: it was about 3 micromolar, which was certainly much lower than the arsenate concentrations (by a factor of about 10,000).
But here's the relevant question: Is 3 micromolar phosphate a lot? Or a little? One point of comparison is the Sargasso Sea, where plenty of microbes survive and make normal DNA. Here, the phosphate concentrations are less than 10 nanomolar - or 300 times less phosphate than the "phosphate-free" media in the GFAJ-1 experiment. At such low phosphate concentrations, some bacteria compensate by removing phosphorus from their lipids - but not from their DNA.
So the Sargasso Sea tells us that some bacteria are capable of making DNA at very low phosphate concentrations. The most plausible explanation is that the bacterium GFAJ-1 can make normal DNA at micromolar phosphate concentrations, and that it also has the ability to tolerate very high arsenate concentrations.
There are numerous other aspects of this study that don't make sense. Why would bacteria from Mono Lake need the ability to substitute arsenate for phosphate in their DNA? Yes, arsenic concentrations are high in Mono Lake. But so are phosphate concentrations, which approach 1 millimolar - or 100,000 times higher than in the Sargasso Sea. Mono Lake has more phosphate available than nearly any other environment on Earth. There is no selective pressure for the evolution of what would surely be a massively complex switch in nucleic acid chemistry from phosphate to arsenate. I can only begin to imagine the structural problems that would be imposed on DNA by this switch, which would change bond lengths between nucleotides, and cause secondary problems with transcription, etc. Then there is the radical suggestion that nucleotide chemistry is stable because might occur in a 'non-aqueous' environment. It's not clear how that could work.
Finally, there's a simple experiment that could resolve this debate: analyze the nucleotides directly. Show a mass spectrum of DNA sequences demonstrating that nucleotides contain arsenate instead of phosphate. This is a very simple experiment, and would be quite convincing - but it has not been performed.
This study lacks any real evidence for arsenate-based DNA; unfortunately these exciting claims are very very shaky.
Update (12/6/2010): Dr. Rosie Redfield has a quantitative discussion of why there's plenty of phosphorus here.
Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF, Webb SM, Weber PK, Davies PC, Anbar AD, & Oremland RS (2010). A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus. Science (New York, N.Y.)
[NOTE: Because this post is now a magnet for spam, commenting has been closed. If you want to leave a comment, please send it to me via e-mail: webeastiesblog (at) gmail.com]
Thanks for the informative post. This is exciting research; it is not surprising that the excitement is running ahead of the evidence. It will be interesting to see how this develops.
Wow! Another sensational piece of bad science published by the big science outlets.
Sounds like there we have it, for now.
Thanks for reading and commenting!
Paulino... I wouldn't go so far as to call this bad science, at least not yet. I am currently writing a post explaining why. Please check back soon!
Thanks for the post Alex. This seems like a pretty straightforward and obvious problem - you'd think a reviewer would have picked up on it. And shame on NASA for all the bullshit hype they trotted this out with. We have a big enough problem with the public perception of science without such obvious blunders.
Thanks, Alex, for providing the chemist's view into the weaknesses of this paper. I've put the microbiological view (also finding many serious flaws) up at my blog, RRResearch. Click on my name below to see it.
Hi Rosie - thanks for your comment.
I definitely encourage you all to go to her blog and read her detailed and thorough review!
While I find the report odd and unlikely, I have to dissagree with your assumptions on two points.
1. You are assuming that all arscnic based compounds behave like their pnosphate counterparts. Failure to react like normal dna is no rigid criteria for the existance of weird dna. Odd conjugates of metals behave in maners that are strange to both organic and inorganic chemists.
2. The second problem is bond angles. Can one really replace phosphate and create a structure that resembles dna. Even a few degrees bond angle per turn would completely change how dna polymerase and dozens of related proteins work. Replacement of any component with normal phosphate dependant processes would make the As chemistry less probable.
there is no conflict between your statements and the argument in the post.
re: #1 - that is the whole point of Alex's argument. phosphate and arsenate compounds should have different chemistry. how could this possibly be more clear? did you read the post?
re: #2 - sure, different bond angles are another reason, among many, that the speculation that arsenate replaces phosphate in DNA of this organism is ridiculous. but this has no relevance whether the arguments presented in the post here are accurate.
I agree with 99% of this analysis, but wouldn't be so quick to zero in on the rate of hydrolysis of the putative As-esters. Ester hydrolysis (of any flavour) is *very* substrate and substitution specific, so it's not insane to image that these are just very slow to hydrolyze.
This is supposed to be Science; Not some political arena where you either Agree or Disagree with findings. As a first, to test whether or not the lifeform is arsenic based, you'd try to reproduce the tests conducted by NASA to see where and why (if it) deviates. Some assumptions on how 'what if' and a lot of guessing on intrinsic parameters only drills holes in this Article.
That means the archaeon is interesting for an entirely different reason: its ability to keep arsenate out of its ATP synthase and its phosphorylases. Inability to do that, followed by hydrolysis, is why arsenic oxides are toxic in the first place.
Great Post! I hope we'll see the results of these controls in a later paper from Wolfe-Simon. It is intriguing that the reviewers didn't bring up these sorts of questions during the review process. Were they caught up in the excitement?
There's a lot to be learned from this organism regardless of whether or not it has arsenate in its backbone.
I propose a very simple way to test the idea of arenate-containing DNA!
Take the DNA of the cells grown on +As/-P and -As/+P medium and apply it to density gradient centrifugation! If you see that the +As one is migrating lower than the -As one you have a point!
I donÂ´t understand that the reviewers let this paper through! Incredible!
I am disappointed in everyone jumping in and screaming 'bullshit' so quickly...
When a news organization (not a peer reviewed journal like Science) over-sensationalizes and misinterprets a study, adds nothing to the scientific conversation, and makes a profit, THAT is bullshit.
When a finding is published, but there are alternatives to the interpretations set forth in the discussion, follow-ups needed to be done, and even flaws in the methodology, that is called "the Scientific method."
Great post! Thank you!
This sounds incredibly interesting but if it's true, this needs to be published as a formal response in Science.
You seem to claim that they did not do a mass spec analysis... It appears they did. Am I mistaken?
"We then used high-resolution secondary ion mass spectrometry (NanoSIMS) to positively identify As in extracted, gel purified genomic DNA (Fig. 2A). These data showed that DNA from +As/-P cells had elevated As and low P relative to DNA from the -As/+P cells. NanoSIMS analysis of the DNA showed that the As:P ratio on an atom per atom basis was significantly higher in the +As/-P versus -As/+P grown cells"
If this is a big blunder (and it's looking more and more like that's the case), it is a great illustration of how science is self-correcting. Mistakes are made in science, the scientific methods allows us to identify and correct those mistakes. How often do we see this in creationist and other pseudoscientific circles? In fact, this is one of the most significant distinctions between real and make-believe science. We should be proud that criticism of this paper has come so quickly and with such force.
As badly as I want this arsenate "second genesis" to be true, I love this rebuttal that much more. Well written. well explained, testable and refutable. Thank you, Kevin, and good luck with your upcoming defense.
Writer man, will you marry me? I promise I won't tell my boyfriend. I've been rebutting this to my classmates and friends all week. But you did a great job. Guess I should read more papers... stupid PhD.
Alex and Kevin: Check out the CBC News article: http://www.cbc.ca/technology/story/2010/12/06/arsenic-microbe-dna-nasa-…
Which is more astounding, the tricks that microorganisms can pull, the oversights of reviewers, or the willingness of the mass media to jump on a hype wagon? I recall seeing a report of this paper as the centerpiece of the New York Times homepage! Following up, I couldn't believe it. It's obvious that reviewers -- and editors -- were not thorough enough. Everyone in the process is overloaded, we can all make mistakes, but headline grabbers like this need special attention. So thanks to Alex Bradley for a much-needed response. I hope we don't go to a system where on-line debate is the real review, but it's salutary in this case, for sure.
Everyone seems to be focusing on DNA. I concur with the post that the backbone is likely P-based.
In my opinion, it's ATP that is more likely to have some As-based biological "work around" or use some other energy storage/transport biology.
It seems to me that a main feature of Eastern Sierra/Basin and Range salt lakes is that they generally cycle between wet and dry. At my level of geochemistry knowledge, I know that this affects mineralization and re-mineralization. Also, as I understand it, life forms are adapted to swings in salt content. (At my level of expertise, this includes brine shrimp, bug larvae, and living, carbon based, "gloppy stuff"; as well as birds, sometimes fish and other higher life forms that can feed on them).
Mono Lake is a special case in that it is large (but with a number of more brackish side sloughs). The city of Los Angeles started diverting its source waters in 1941 and drove the lake towards near dryness by the early 1990's. Since then, forced, I believe by air pollution regulations and dust bowl like conditions, water has been put into the lake. It is my understanding that the lake is still refilling up to a mandated level. See:http://www.swrcb.ca.gov/waterrights/water_issues/programs/mono_lake/doc…
So, wouldn't bacteria in this lake be in the process of adapting back to wetter, less salty conditions?
I guess I am having trouble understanding how the control in the experiment doesn't hold up under scrutiny? I thought they had a negative control plate (-A/-P) that had the same (low phosphate) growth media as the -A/+P plate and the +A/-P plate, and those cells didn't grow? Shouldn't this imply that it requires at least a supplement of arsenate to partially compensate for low phosphate?
I do, however, agree with your suggestion that the scientists should do a second control with the gel.
Also - what is ICP-MS? Is this mass-spec methodology also insignificant within the parameters of this experiment? If it says that ICP-MS was used on the nucleic acid fractions, does this also imply that this mass spec wasn't specific enough?
Also: "In contrast to the models, these As-O and As-C
distances are consistent with that reported from the solved
crystal structure of DNA for the analogous structural position of P relative to O and C atoms (Fig. 3A) (16, 17). Therefore, our X-ray data support the position of arsenate in a similar configuration to phosphate in a DNA backbone"
Is this also to be ruled out?
I sort of feel like other scientists should be attempting to reproduce this experiment/ try out new methods before discounting it as a media ploy.
First, a comment about a small group of commentors I've seen commenting on many of the stories about this discovery (if that's what it is): why is it that some people accuse every single scientist of being driven first and foremost by greed? There must be hundreds of thousands of scientists all over the world, each an individual. Are some of those scientists/ motives open to question? -- sure. Consider, for instance, scientists on the payroll of companies involved in fossil fuels or tobacco companies. And, for balance, consider those on the payroll of activist "green" groups and organizations.
Having known a few scientists -- I'm not one myself -- I've always accepted that while there undoubtedly are a few driven by money and/or questionable agendas, the great majority are motivated by a desire to do real science.
Anyone inclined towards a contrary position needs to keep in mind three concepts, one from psychology, two from logic. From psychology, projection. From logic, hasty generalization and ad hominem arguments. I've seen countless examples of all three, often combination with one another.
Enough about that.
Born in 1951, I've been interested in space and the possibility of alien life since as far back into my childhood as I can remember, an interested greatly sharpened when Dad took me outside to watch Sputnik soar overhead. (Okay -- no, we didn't see Sputnik, but its booster, which was orbiting behind it. But I didn't know it that magical night, and neither did Dad, until I told him years later, when I found out myself. I imagine I would have every bit as enthralled had I known what I was actually seeing.) Superhuman was my favorite comic hero. (Hey, super-powers AND alien! What more could one ask???)
It probably surprise no one for me to say I keenly hope there is alien life elsewhere in the Universe, even if it's so simple it's to bacteria what bacteria is to a genius. Of course, intelligent aliens (benevolent or peaceful ones) would be nice, even if all we got were a radio or laser signal.
If an earth-bound discovery leads us to broaden the definition of "life" -- great. But I had questions almost from the very start, not because of scientific questions about the researchers' methodology and interpretation -- those arose a day or two later, when I started seeing items such as this blog entry -- but because while much of the mass media were reporting this as a discovery of another life form, a few lonesome voices were pointing out that such a conclusion made good headlines -- but not good science.
In any chase, even if it turns out over the long term that our current understanding of the requirements for life -- life anywhere -- is correct, there remain almost limitless possibilities, particularly considering that astronomers have just upped the estimate of the number of stars by three and that they've also taken another good, long, ard look at red dwarfs as a class of star that conceivably could support life, if not in conditions that would entice us to vacation on a planet orbiting a red dwarf in its "Goldilocks zone."
There *is* that pesky spped-of-light maximum speed limit that hampers our search, darn it. . . .
Bizde en iyi yerlere gelmek icin bir caba icindeyiz tabiki bunun zorlugunu yasamaktayiz ama bir yola ciktik ve bunu basarmaya Ã§alismaktayiz bakalim nasÄ±l olucak istedigimize ulasabilcekmiyiz inanin boyle cok zor oluyor ama mecburuz napalim evet hayirlisi .
I read the paper but now I have not got it within my reach.
So, Correct me if I am wrong.
Did they measure As:C and P:C ratios from DNA extracts using NanoSIMS? And did they get a result that As+/P- cells had lower P:C ratio than As-/P+ cells? If both of these cell types had DNA with P-backbone then would the P:C ratio also be the same? Or was there more C-including contaminants in As+/P- cell extracts than in As-/P+ extracts? Or is there also extra P associated with DNA extracts of As-/P+ cells as there might be extra As in extracts of As+/P-.
And can it really be that they did not wash DNA pellet with ethanol? I have always thought that this is a step which always has to be carried out when exrcating DNA?
Thanks for posting such a clearly-written commentary about possible problems w/the research. I really appreciate it. The amount of hype implying discovery of "alien" life originating right here on earth was bothersome. It is good to read a calmer and cogent response that puts the matter into better perspective.
Correct me if I'm wrong, but shouldn't DNA in a phenol:chloroform extraction that separates DNA and RNA be in the *organic*, not aqueous phase? Sure, some of the DNA may have dissolved in the aqueous phase as it was being purified, but if there was arsenic-based DNA in the sample, some would have remained intact, safely out of the way of water, in the phenol layer.
On top of that, not all phenol:chloroform preps are water-saturated. So where's your water coming from in that case?
I'm personally not convinced of the findings, either, but this, in my humble opinion, is not the reason to doubt it. Then again, I'm just a humble undergrad molecular bio major researching this for her class assignment, what would I know?
Forgot two links to back my opinions. I work with this stuff daily, so I have some idea of what I'm talking about, even if I don't have a degree yet.
Okay, so my brain doesn't work too well this late at night - the phenol should be water saturated at all times, and the DNA should be in the phenol phase. Haha, stupid me.
In that case, this is yet more evidence of the strangeness that happened with this particular paper, in addition to referencing papers incorrectly and so forth...
Really need to be careful, treading these waters.
My students are having a field day with the events as they unfold. We began a discussion the day the news came out and listed all the questions we would ask the researches, given the opprtunity. Now we discuss what is happening in the blogs. They are quite amazed to discover the real scientific process in action. Great postings....
Nepheliad (#31,32,33) -- in organic extractions of DNA, the DNA remains in the AQUEOUS phase -- you use water-saturated phenol to prevent it from sucking some water out of the buffer. As it says in the openwetware phenol link you cited ( http://openwetware.org/wiki/Phenol ):
"To purify DNA, water saturated phenol (pH 7.9) or phenol chloroform is added to approximately equal amounts of water or buffer containing DNA and vortexed. Brief centrifugation brings the phenol to the bottom, leaving purified DNA in the water solution. ... Protein typically is evident as a white precipitate forming at the boundary between the phenol and water phases."
This is supposed to be Science; Not some political arena where you either Agree or Disagree with findings. As a first, to test whether or not the lifeform is arsenic based, you'd try to reproduce the tests conducted by NASA to see where and why (if it) deviates...
This is supposed to be Science; Not some political arena where you either Agree or Disagree with findings.