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 visitors in 2012: We've just submitted a manuscript to Science reporting the results of our unsuccessful attempt to replicate the key findings of this work. The manuscript will be publicly available on the arXiv server beginning Feb. 1 2012.
Newer note to new readers: See also my new (Dec. 16) critique of the authors' response to these and similar criticisms.)
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). 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. (A researcher in my lab had a better explanation - I've put it in the Comments below.)
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 assuming that a phosphorus-starved cell uses half of its phosphorus for DNA and the rest for RNA and other molecules, 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). Many bacterial cells produce BHP when their carbon/energy supply is good but other nutrients needed for growth are in short supply. Cells grown with phosphate 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 added 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.