Arsenic-based DNA in bacteria? NOT!
Just as I predicted, the Darwinists make a big and misleading announcement and then the fizzle-out comes...
[guest post: Alex Bradley, PhD] Arsenate-based DNA: a big idea with big holes
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. Iwill be postinghave posted my personal and less-technical take on the whole thingsoon, so stay tunedas 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.) PMID: 21127214
~~~~~~
Rosie's update:
Arsenic-associated bacteria (NASA's claims)
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.) PMID: 21127214
Note to new readers: I wrote this post on Saturday Dec. 4, mainly to clarify my own thinking. I didn't expect anyone other than a few researchers to ever read it. Since then I've made a few minor corrections and clarifications (typos, decimal places, cells not cfu), but I haven't changed anything significant. Please read the comments - they contain a lot of good scientific thinking by other researchers.
Here's a detailed review of the new paper from NASA claiming to have isolated a bacterium that substitutes arsenic for phosphorus on its macromolecules and metabolites. (Wolfe-Simon et al. 2010, A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus.) NASA's shameful analysis of the alleged bacteria in the Mars meteorite made me very suspicious of their microbiology, an attitude that's only strengthened by my reading of this paper. Basically, it doesn't present ANY convincing evidence that arsenic has been incorporated into DNA (or any other biological molecule).
What did the authors actually do? They took sediment from Mono Lake in California, a very salty and alkaline lake containing 88 mg of phosphate and 17 mg of arsenic per liter. They put the sediment into a similarly alkaline and hypersaline defined medium containing 10 mM glucose as a carbon source, 0.8 mM NH4SO4 as a nitrogen and sulfur source, and a full assortment of the vitamins and trace minerals that might be needed for bacterial growth. Although this basic medium had no added phosphate or arsenate, contamination of the ingredients caused it to contain about 3 µM phosphate (PO4) and about 0.3 µM arsenate (AsO4) as. For bacterial growth it was supplemented with arsenate or phosphate at various concentrations.
The interesting results came from sediment originally diluted into medium supplemented with the highest arsenate concentration they initially tried (5 mM) but no phosphate. Over the course of several months they did seven tenfold dilutions; in the sixth one they saw a gradual turbidity increase suggesting that bacteria were growing at a rate of about 0.1 per day. I think this means that the bacteria were doubling about every 10 days (no, every 7 days - corrected by an anonymous commenter).
After one more tenfold dilution they put some of the culture onto an agar plate made with the same medium; at least one colony grew, which they then inoculated into the same defined medium with 5 mM arsenate. They gradually increased the arsenate to 40 mM (Mono Lake water contains 200 µM arsenate). Descendants of these cells eventually grew in 40 mM arsenate, with about one doubling every two days. They grew faster if the arsenate was replaced by1.5 mM phosphate but grew only about threefold if neither supplement was provided (Fig. 1 A and B, below). The authors misleadingly claim that the cells didn't grow at all with no supplements.
In Fig. 1 (below), the correspondence between OD600 (Fig. 1 A) and cells (Fig. 1 B) is not good. Although the lines in the two graphs have similar proportions, OD600 is plotted on a linear scale and cells/ml on a log scale (is this a shabby trick to increase their superficial similarity?). OD600 in arsenate medium was almost as high as that in phosphate medium, but the number of cells was at least tenfold lower. And the OD in arsenate continued to increase for many days after the cells has leveled off. I suspect most of the continuing growth was just compensating for cell death. It would be interesting to test whether the cells were scavenging phosphate from their dead siblings.
The authors never calculated whether the amount of growth they saw in the arsenate-only medium (2-3 x 10^7 cfu/ml) could be supported by the phosphate in this medium (or maybe they did but they didn't like the result). For simplicity I'll start by assume that a phosphorus-starved cell uses half of its phosphorus for DNA and the rest for RNA and other molecule, and that the genome is 5x10^6 bp. Each cell then needs 1x10^7 atoms of phosphorus for DNA, and 2x10^7 for everything. The medium is 3.1 µM phosphate, which is 3.1x10^-6 moles per liter. Mutiply by Avogadro's number (6.02x10^23 atoms per mole) and we have 1.9x10^18 atoms of phosphorus per liter, or 1.9x10^15 per ml. Divide by the phosphorus requirement of each cell (2x10^7) and we get 9.5 x 10^7 cells per ml. This value is just comfortably larger than the observed final density, suggesting that, although these bacteria grow poorly in the absence of arsenate, in its presence their growth is limited by phosphate. (Note: This calculation originally dropped a decimal point. I've changed it a bit and corrected the error.)
Under the microscope the bacteria grown with arsenate and no added phosphate (Fig. 1 C) look like plump little corn kernels, about 1 µm across and 2 µm long. They contain many structures (Fig. 1 E) which the authors think may be granules of the wax-like carbon/energy storage material polyhydroxybutyrate (PHB). Bacterial cells produce this when their carbon/energy supply is good but other nutrients needed for growth are in short supply. Cells grown with phospate and no added arsenate are thinner and lack the granules (Fig. 1 D). The authors used 16S rRNA sequencing to identify this bacterium as belonging to the genus Halomonas, a member of the gammaproteobacterial order Oceanospirillales. Members of this group are diverse but not known to have any uniquely dramatic features.
According to an interview with the first author, this research was motivated by a desire to show that organisms could use arsenic in place of phosphorus. The two atoms have very similar chemical properties, but bonds with arsenic are known to be much less stable than those with phosphate, so most researchers think that biological molecules containing arsenic rather than phosphorus would be too unstable to support life. Thus the authors wanted to show that the bacteria had incorporated the arsenic in places where phosphorus would normally be found. They used several methods, each involving a low-tech preparation of cell material and a high-tech identification of the atoms present in the material.
First they collected the bacteria by centrifugation, washed them well, and precisely measured the fraction of arsenic and phosphorus (as ppb dry weight, Tables 1 and S1). Cells given only the arsenate supplement contained about 10-fold more arsenic than phosphorus (0.2% arsenic and 0.02% phosphorus) and cells given only the phosphate supplement had 0.5% phosphorus and only 0.001% arsenic.
The authors argue that the arsenate-grown cells don't contain enough phosphorus to support life. They say that typical heterotrophic bacteria require 1-3% P to support life, but this isn't true. These numbers are just the amounts found in E. coli cells grown in medium with abundant phosphate. They are very unlikely to apply to bacteria growing very slowly under phosphate limitation, and aren't even true of their own phosphate-grown bacteria (0.5% P). The large amount of PHB in the arsenate-grown cells would have skewed this comparison - PHB granules are mainly carbon with no water, and in other species can be as much as 90% of the dry weight of the cells. Thus their presence only in arsenate-grown cells could depress these cells' apparent phosphate concentration by as much as 10-fold.
The authors then grew some cells with radioactive arsenate (73-As) and no phosphate, washed and dissolved them, and used extraction with phenol and phenol:chloroform to separate the major macromolecules. The protein fraction at the interface between the organic and aqueous phases had about 10% of the arsenic label but, because the interface material is typically contaminated with liquid from the aqueous phase, this is not good evidence that the cells' protein contained covalently-bound arsenate in place of phosphorus. About 75% of the arsenic label was in the aqueous (upper) fraction. The authors describe this fraction as DNA/RNA, but it also contains most of the small water-soluble molecules of the cell, so its high arsenic content is not evidence that the DNA and RNA contain arsenic in place of phosphorus. The authors use very indirect evidence to argue that the distribution of arsenic mirrors that expected for phosphate, but this argument depends on so many assumptions that it should be ignored.
(They also measured the absolute amounts of arsenic and phosphorus in the supernatant fraction - surprisingly, no arsenic (<20 ppb) was detected in the fraction from arsenate-supplemented cells, although the fraction from phosphate-grown cells had 118 ppb! See Table S1.)
They especially wanted to show that the cells' DNA contained arsenic in place of phosphorus, so they gel-purified chromosomal DNA from cells grown with arsenate (lane 2) or with phosphate (lane 3), and measured the ratio of arsenic to carbon by mass spectrometry. The numbers at the bottom give these ratios (the legend says 'multiplied by 10^-6 but they surely mean 'multiplied by 10^6').
As expected, this ratio was very low for the phosphate-grown cells (6.9x10^-6), but it was only twofold higher for the arsenate-grown cells (13.4x10^-6). Normal DNA has one phosphorus atom for each ten carbons (P:C = 10^-1), so the arsenate-grown ratio is only about one arsenic atom per 10,000 phosphorus atoms (i.e. one per 5 kb of double-stranded DNA). A 2x10^6 bp genome would contain 4x10^6 atoms of phosphorus, so if all this arsenate was really covalently in the DNA, each genome would only contain about 400 atoms of arsenic. And a phosphate-grown genome would contain 200!
Could 400 atoms of arsenate per genome be due to carryover of the arsenate in the phenol-chloroform supernatant rather than to covalent incorporation of As in DNA? The Methods describes a standard ethanol precipitation with no washing (and no column purification which would have included washing), so I think some arsenate could easily have been carried over with the DNA, especially if it is not very soluble in 70% ethanol. Would this arsenate have left the DNA during the gel purification? Maybe not - the methods don't say that the DNA was purified away from the agarose gel matrix before being analyzed. This step is certainly standard, but if it was omitted then any contaminating arsenic might have been carried over into the elemental analysis.
Failure to purify the DNA away from the agarose would also compromise their elemental analysis in other ways, since much of the carbon in the purified 'DNA' would have been from the agarose. The authors did do the same elemental analysis on a gel slice with no DNA in it, a control that only makes sense if they didn't purify the DNA. Not purifying away the gel might affect the arsenate-grown DNA more because the band contains less DNA; this would explain why this excised DNA has 3.5-fold lower ratio of phosphorus to carbon than the phosphate-grown DNA, a difference that is certainly not explained by its very low arsenic content.)
(Might they have not presented assays using properly purified (washed) DNA because these turned out to not have any arsenic? Am I just paranoid?)
Finally, the authors examined the chemical environment (neighbouring atoms and bonds) of the arsenic in the cells using synchrotron X-ray studies. This is over my head, but they seem to be trying to interpret the signal as indicating that the environment of the arsenic is similar to that of phosphorus in normal DNA. But the cellular arsenic being in DNA can't be the explanation, because their DNA analysis indicated that very little of the cellular arsenic purifies with the DNA. The cells contained 0.19% arsenic (1.9x10^6 ppb), but the DNA only contained 27 ppb arsenic.
Bottom line: Lots of flim-flam, but very little reliable information. The mass spec measurements may be very well done (I lack expertise here), but their value is severely compromised by the poor quality of the inputs. If this data was presented by a PhD student at their committee meeting, I'd send them back to the bench to do more cleanup and controls.
There's a difference between controls done to genuinely test your hypothesis and those done when you just want to show that your hypothesis is true. The authors have done some of the latter, but not the former. They should have mixed pregrown E. coli or other cells with the arsenate supplemented medium and then done the same purifications. They should have thoroughly washed their DNA preps (a column cleanup is ridiculously easy), and maybe incubated it with phosphate buffer to displace any associated arsenate before doing the elemental analysis. They should have mixed E. coli DNA with arsenate and then gel-purified it. They should have tested whether their arsenic-containing DNA could be used as a template by normal DNA polymerases. They should have noticed all the discrepancies in their data and done experiments to find the causes.
I don't know whether the authors are just bad scientists or whether they're unscrupulously pushing NASA's 'There's life in outer space!' agenda. I hesitate to blame the reviewers, as their objections are likely to have been overruled by Science's editors in their eagerness to score such a high-impact publication.