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I've been saying that researchers shouldn't invest the time and resources needed to test Wolfe-Simon et al's claims, because of the vanishingly small probability that they are correct. But I'm having second thoughts, because the most important claims can, I think, be very easily tested. So I've just sent an email off to GFAJ1samplerequest@gmail.com (thanks Dave Baltrus, @surt_lab, for tweeting this address), asking for information on how to obtain the bacterial strain GFAJ-1.
The main questions to answer are:
Q. 1. Is the approximately tenfold growth difference between +As/-P and -As/-P in Figure 1B due to the cells' use of As in place of P in DNA, RNA and other biomolecules?
Q. 2. Does DNA purified from cells grown with limiting P and abundant As contain significant amounts of covalently incorporated As?
Both of these questions can be answered by straightforward experiments. However a microbiologist like myself would need to work with someone (a chemist?) who could use a mass spec to assay As and P in the reagents, media and DNA. I'm assuming this is very straightforward, but perhaps readers can set me straight if I'm wrong.
I'll consider Q. 2 first because it initially seemed easiest.
1. Make culture media with 40 mM arsenate and varying levels of phosphate (at least 0, 3 and 1500 µM). The recipe for the base of 'AML60 salts' is available here; unfortunately this reference does not give the components of the 'trace metals stock' it uses, nor does the reference it cites. Don't worry about any phosphate contamination of the medium.
2. Inoculate the GFAJ-1 cells into the different media and culture them in screw-top vials at 28 °C in the dark. The Methods don't say anything about mixing, so probably stationary culture with occasional mixing is OK. I could turn down the temperature of one of my new/old incubators if we were to do this.
3. Monitor the culture growth by counting a defined volume of the cells under a microscope (a hemocytometer is useful for this). The cells will probably double every day or so.
4. Once the cells have stopped dividing, collect them by centrifugation and wash them several times with a simple solution such as PBS.
5. As a control for arsenic contamination, add to arsenic medium an equal number of E. coli cells that have been grown in medium without arsenic, and let them sit for a couple of hours. Then collect them and wash them along with the GFAJ-1 cells.
6. Mix the cells with 50 mM Tris 10 mM EDTA (no more than 10^9 cells per ml) and lyse them by adding SDS to 1%. Add RNase A (10 mM) and incubate at 37 °C for 20 minutes to degrade the RNA. Extract the lysed cells with twice with phenol and twice with phenol:chloroform.
7. To the aqueous phase add NaCl (150 mM) and ethanol to 70%. Collect the chromosomal DNA by spooling it onto the sealed tip of a glass Pasteur pipette. Wash the DNA by rinsing the tip with 70% ethanol, and let the DNA air-dry on the tip.
8. Dissolve the DNA in TE (10 mM tris 1 mM EDTA), aiming for a concentration of about 200 µg/ml, based on the number of cells you had in each prep.
Now further purify each DNA using all three of the following methods. Each method has different advantages and is likely to remove different kinds of contaminants, and I've ordered them by the amounts of DNA they can handle. (Perhaps you should first ask your chemist how much DNA is needed for mass spec analysis.):
9. Repeat the spooling precipitation: Add NaCl and ethanol, and again spool the DNA as it is driven out of solution by the ethanol. Spooling works well with chromosomal DNA because the DNA concentration is usually high and the DNA fragments are long. It is preferable to centrifugation because small ethanol-insoluble molecules that would pellet with the DNA are left behind.
10: Purify the DNA on a spin column: These columns will bind about 10 µg of DNA, and the bound DNA can then be washed by repeatedly passing an alcohol solution through the column. The DNA is then eluted by washing TE through the column.
11. Purify the DNA by gel electrophoresis: Load some of the DNA into the well of an agarose gel, and electrophorese it until the DNA has migrated several cm into the gel. Use a small enough amount of DNA and a large enough gel and well that the DNA runs as a clean band and not as a smear. Cut out the band and purify the DNA away from the agarose and gel buffer using a spin-column kit.
12. Give the DNAs to the chemist for mass spec analysis. Be sure to have DNA from bacteria other than GFAJ-1, grown without arsenic, with and without soaking the cells in arsenic medium before DNA purification.
This may sound like a lot of work, but steps 4 - 12 can easily be completed for 3-6 DNAs in a single day. If the DNA from cells grown in As plus limiting P contains no more arsenic than the other DNAs, then the answer to Q. 2 is "No".
Now I'll consider Q. 1. It's just like the first steps of Q. 2, but with the mass spec done on the media at the beginning rather than on the DNA at the end.
1. Find out how much phosphate contamination to expect in ultra-pure versions of the reagents needed to make the AML60-based culture media - your chemist collaborator should have access to this information. Use this information to calculate how much P to expect in the basic medium with and without As. You could skip this step, but it would be prudent to check this in advance.
2. Order ultra-pure reagents and make the media, or have your chemist collaborator make the media. You want each of the following: plain glucose media with no added As or P, media with 3 µM P and no As, media with 40 mM As and no P, media with 3 µM P and 40 mM As, and media with 1500 µM P and 40 mM As.
3. Have the chemist assay the media for As and P by mass spec.
4. Inoculate the GFAJ-1 cells, first washing them to remove traces of whatever medium they've been growing in/on.
5. Monitor the culture growth by counting the cells under a microscope. The cells in cultures with added P will probably double every day or so.
6. Once the cells have stopped dividing, plot cell numbers as a function of time for each medium. If final cell density is proportional to the amount of P in the medium, then the answer to Q. 1 is "No".
Maybe have the chemist assay the spent media from the low-P cultures to see how the P concentration has changed.
Of course, for either experiment the most important practical question is 'Could these results be published?' I think they could. The best plan would be to have several independent labs do the same test and publish jointly - we'd have more impact and all get our names on a publication. On the other hand, we all have more important things to do...
Something that concerns me about any studies of a putative As-DNA bacterium (not that I give the study in question much credence, mind) is the apparent instability of As-DNA and what this means to analyses.
ReplyDeleteIn other words, if there *were* an Arsenic-based lifeform, I would expect the DNA to have crosslinked proteins or glycosides to protect the arsenic from water-mediated degradation. What will this mean for analysing the DNA using strong organic solvents, rigorous desalting, etc, which might remove whatever keeps the DNA stable?
I'm not offering a "you can't test it so it must be true", but rather trying to suggest that you close off any avenues of excuse if/when you disprove the "Arsenic Life" notion.
All this comes from my confidence that, actually, the "requirement" for CHNOPS is an artefact of terrestrial life. Somewhere out in the universe there may indeed be arsenic-based life. Life would find a way to protect even an unstable molecule such as As-DNA from destruction, just as it does with countless other unstable polymers. I just don't think that's what we've seen with this unfortunate media-fuelled mixup.
Hmmm... probably wouldn't wash the cells in PBS, of all things... maybe use TBS? Why not just use a standard kit for the DNA extraction instead of the laborious phenol/chloroform? Quicker and cleaner. Then run the gel, excise, purify again.
ReplyDeleteI would probably digest the DNA and look for P or As nucleotides, looking at the whole DNA will be a mess. Stability will not be a problem, as we all know now that As-nucleotide hydrolysis is sterically hindered....;-P
Even if they aren't you should then see a higher nucleoside/nucleotide ratio in the As condition.
Also, someone should do an autoradiography of the gel.
Justin Cayce
PS and unrelated: Nobody seems to have mentioned that the EXAFS is pretty irrelevant as arsenate esters will form sponaneously under those conditions (dessication in the presence of arsenate).
I like your suggestions for further research to falsify the claims put forward by Wolfe-Simon et al., Rosie, but frankly, why bother? Everybody knows their claims are not supported by any evidence. File this in the same folder as "polywater", "cold fusion" and "memory of water" publications that have appeared in top tier journals previously and just move on with your own research which holds so much more promise.
ReplyDeleteBack in my undergrad days I used to run GC for practice. If all you need is a printout that is really quite easy. I reckon you could just go to your local university and ask the OChem TA if he wouldn't mind adding your As-DNA to a class run of GC. It takes about 15 minutes to take care of the whole thing. The interpretation is a different story of course, but that can be done at leisure or put up on your blog and someone (not me been AGES since I have done this stuff) would certainly have the knowledge to help you read it.
ReplyDeleteI like the idea and it should be something that could pretty easily be knocked out in just a few days - week at the most.
I agree with the previous comment from 'Anonymous'. A couple of rounds of purification with the standard kits should be easily enough to demonstrate the difference between As-DNA and the regular variety, if there is one. Add extra washes to the spin column protocols if you like. It's quite amazing that the original paper was accepted without requiring this experiment, since it should be dead easy to do.
ReplyDeleteIf the chromosomal As-DNA is not stable under the conditions of the purification protocol, that should be quite straightforward to control for by running samples from the different stages on agarose gel.
@QStel--
ReplyDeleteIt's easy to say something isn't important. But think of all the time and effort saved if replication experiments were routinely done in fields such as medicine. I think it's too bad that people don't seem interested (or can't be b/c of the whole pressure to publish positive results).
Cold fusion is a bad example. A lot of people tried replicating it and got no dice. So we file it under "cool idea if true, but it isn't."
There seem to me to be two problems with trying to estimate the arsenate content of arsenate-grown GFAJ-1 DNA. One already canvassed is that there might be arsenic somehow adhering to the partially purified DNA. This can be reduced by further purification steps. The other side of the issue is that as you purify the DNA you may continue to lose arsenic because the unstable arsenyl DNA diesters are lost from the DNA by hydrolysis.
ReplyDeleteAnother way to estimate the fraction of phosphate replaced by arsenate in DNA that avoids these issues is to obtain quickly a partially purified sample of DNA. Then allow the arsenyl esters to completely hydrolyse by incubating in a buffer such as TE (1) (with EDTA to chelate Mg2+ etc so that any contaminating DNAses cannot function). The end result of this would be DNA containing nicks wherever an arsenate was present. Provided the nicks are sufficiently far apart the DNA should hold together because of the pairing of the overhangs, A good example of DNA holding together despite nearby nicks are the cohesive (cos) ends of lambda phage. These are 12 base overhangs that anneal/base pair together in packaged lambda DNA. These annealed ends can survive gel electrophoresis so the usual technique to dissociate the ends is to heat them at 65° before electrophoresis. Now the data of Wolfe-Simons et al suggested that their DNA contained about 0.04 mole arsenic per mole of phosphate. If this was left to hydrolyse completely then the resulting DNA would contain a nick, on average, every 25 bases. This would mean an average overhang of about 12 nucleotides like the lambda cohesive ends. So heating the now arsenate-free DNA to denature it followed by electrophoresis (eg by a previous generation denaturing sequencing gel) would show the size distribution of the DNA and thus allow the arsenate content of the original DNA to be estimated. The denatured DNA could be end labelled with 32P ATP and polynucleotide kinase (or fluorescently labelled with T4 RNA ligase) to estimate the size distribution of smaller quantities of DNA. It seems advisable not to restrict the DNA purification to only high molecular weight DNA because more highly nicked DNA may be lost during such a procedure. GFAJ-1 DNA grown in the absence of arsenate should be an appropriate control.
DNA is constantly monitored and repaired in vivo so it would be no surprise that the nicks generated in DNA containing mis-incorporated arsenic will be repaired. Thus there may be nicks present in the DNA in vivo. All the more reason not to bias results by purifying high molecular weight DNA. This brings up a related issue. While double stranded DNA repair is well known, what about repair of RNA? It seems unlikely that a nick in mRNA could be repaired but mRNAs are short lived anyway. On the other hand ribosomal RNAs and the ribosomes themselves are more long lived and have substantial secondary structure brought about by base pairing. Could a ribosome stay intact and function with a substantial number of nicks? It is worth noting that the gels of nucleic acids obtained from GFAJ-1 grown in the presence or absence of arsenate showed that there was little or no ribosomal RNA present in the arsenate-grown nucleic acid preparation. A possible explanation for this is that the highly nicked ribosomal RNA was denatured during the phenol treatment and the short RNA oligos were lost in subsequent purification steps. An analysis of small RNA molecules of the arsenate-grown cells should be revealing.
(1) Room temperature or higher should be fine. Remember DNA survives PCR conditions but if you heat at higher temperatures make sure the DNA doesn't depurinate because of slightly acid conditions. This is because the pH of Tris buffers ( and some others) can drop several pH units when heated to 90°-100°.
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ReplyDelete