Wolfe-Simon et al.'s responses to my comments

This is the first of three more #arseniclife blog posts; the others will consider possible experimental tests of the claims and some evolutionary issues that have been overlooked.

The brief Letter to Science that I composed here (with help from some readers) was one of the eight published today.  The Letter was converted to a 'Technical Comment' by the Science Editorial Office, I guess because it contained technical comments, and had one-paragraph peer reviews from 5 (!) reviewers.  I think Science must have sent all the submissions to the same group of reviewers, who gave each a very brief review.

The Letter made three main points, and I'll treat the authors' responses to each in turn:

1. Because one or more of the reagents used for the culture media were contaminated with phosphate, the growth attributed to arsenic is better explained by growth on phosphorus.  I and several other commenters pointed out that the 3 µM PO4 contaminating most of the media they analyzed would be sufficient to fully explain the observed increase in number of cells.  (My calculation is explained at the end of this post.)

The authors disagree, pointing out that the cells contained less phosphate per total dry weight than needed for growth.  But their dry weight measurements were inflated by the high content of the storage hydrocarbon polyhydroxybutyrate, which contains no phosphate, so this analysis is not valid.

The authors also point out that the cells grew on medium supplemented with As (+As/-P) but not on medium supplemented with neither As nor P.  The graph below is their Fig. 1B, and it shows that the +As/-P culture reached a tenfold higher cell density than the -As/-P  culture.  They say that both media contained the same  ~3 µM PO4 contamination, so the difference in growth must be due to use of As in place of P.  If that were indeed the case, we would expect the cells' DNA and RNA to consist of about 90% As bonds and 10% P bonds, which is certainly not supported by the elemental analyses.

Two other explanations are much more likely.  First, because the bacteria have evolved in an arsenic-rich lake, they might be dependent on arsenic for some metabolic process.  However the cells grew fine in medium with added phosphate.  A perhaps more likely explanation is that the medium used for the -As/-P control did not contain the same level of phosphate contamination.  The data beside the graph in the above figure shows the reported phosphate contamination of different batches of media and solutions.  One batch of medium had no detectable P contamination (< 0.3 µM), despite the authors 'assertion that all media were made up from a stock containing 3 µM P.  This implies that either the media was variably contaminated or the P measurements were unreliable.  The paper does not indicate which batch of medium was used for any given experiment, and the actual source(s) of the P contamination have not been identified (or even sought). 

2. The DNA was not properly purified before gel electrophoresis.  The Methods stated that after extraction with phenol and phenol:chloroform the DNA and RNA was precipitated with ethanol and the pellet resuspended in sterile water.  I pointed out that such organic extractions do not remove any substances such as simple salts that are more soluble in water than in phenol or phenol:chloroform, and that such substances are often less soluble in 70% ethanol and thus would contaminate the pellet.

The new response repeats the authors' earlier claim that phenol and phenol:chloroform extractions remove arsenate and other 'impurities' from the aqueous phase; they provide no citation or experimental evidence to support this statement.  These extractions remove lipids into the organic phase and denature proteins, which form insoluble aggregates at the interface, but I know of no evidence that they remove small hydrophilic molecules such as arsenate.  I've asked other molecular biologists about this and they agree.

The response now says that the pellet from the ethanol precipitation was washed with 70% ethanol before being dried and resuspended in water for further analysis.  This will have removed contaminants that had remained dissolved in the alcohol supernatant, but may not have removed any insoluble material that was pelleted with the nucleic acids.

3.  The DNA was not purified away from the agarose gel before elemental analysis.  After the ethanol-precipitated DNA was resuspended in water it was run in an agarose gel.  Gel electrophoresis is one of the standard ways of obtaining highly purified DNA; it works partly because most contaminants have different electrophoretic mobilities in agarose than DNA does.   But it's essential to then separate the DNA from the agarose that surrounds it.  The authors didn't do this, although it's a standard lab procedure that can be done in 10 minutes with a simple column kit stocked by most labs.  Instead they analyzed the DNA together with the agarose slice it was embedded in.  Because the gel slice contained only about 1 µg of DNA and about 100 mg of 1% agarose gel, it would have contained about 1000-fold more agarose than DNA and about 300-fold more buffer salts than DNA.  Any contaminants in the agarose or in the buffer, or any contaminants introduced with the DNA, would thus have shown up in the elemental analysis.

The authors provide no explanation for leaving the DNA in the agarose.   Instead they just say "Analysis of DNA separated from agarose would be a useful future experiment...".  They follow this with a brief discussion of how their control for the DNA-in-gel analysis may not have been very good, but without in any way admitting that their analysis was flawed.

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Overall, the most striking aspect of the authors' formal response is that they never admit to having made any mistakes or having done anything badly.  This is a bit disconcerting, given how many concerns were raised.