Field of Science

First evidence refuting Wolfe-Simon et al.'s results

I belatedly realized that the data I posted yesterday are quite important, in that they contradict the growth results in Figure 1 of Wolfe-Simon et al.'s paper.

Here's their Fig. 1B (growth curves based on cell counts), annotated with a table of the levels of phosphate and arsenate they found in their media.

Wolfe-Simon et al. used this graph as evidence that the GFAJ-1 bacteria could use arsenic for growth, in place of phosphorus.  They claimed that growth in the 'added AsO4' medium must be due to the cells using arsenic because (1) this -P/+As medium had no added phosphate, and the small amount of phosphate contaminating most batches was insufficient to support this growth and (2) the cells did not grow in the plain medium (-P/-As), to which neither phosphate nor arsenate had been added.

In my blog and in my published Technical Comment I argued that the ~3 µM phosphate contaminating the medium was indeed sufficient to support the observed growth of ~ 2 x 10^7 cells/ml.  The calculation underlying this argument is shown below.  Given the uncertainty in genome size and in the % of cellular P needed for DNA, my new results nicely support this argument - adding 3 µM phosphate to the -P medium supported growth to 1.7 x 10^7 colony-forming units/ml (equal to at least 1.7 x 10^7 cells/ml).
My new results also contradict Wolfe-Simon et al.'s observation that cells could not grow under limiting phosphate if arsenic was not provided.  No arsenic was added to my low-phosphate media, but the cells grew just fine.  I think that GFAJ-1 might have failed to grow in Wolfe-Simon et al.'s batch of -P/-As medium because this batch contained much less phosphate than the other media.  The levels of phosphate contamination they detected (red boxed area above) were very unpredictable (3.7 µM in one batch, <0.3 µM in another, and 7.4 µM in the wash solution), and the Methods don't say that all cultures in this figure were made with the same batch.

I've now set up some new cultures, testing whether the presence of 40 mM arsenate affects growth in media with no added phosphate or with 3 µM or 1.5 mM phosphate.  I hope the sudden introduction of 40 mM isn't too big a shock to the cells; they've been growing without any arsenate for the past two months.
In other progress, the Research Associate has obtained the 16S rRNA sequence of the strain I'm working with.  It exactly matches the sequence of GFAJ-1 in GenBank, so now we're sure these are the right cells.  This confirmation is especially important since the growth properties I'm finding for GFAJ-1 don't match those reported by Wolfe-Simon et al.

8 comments:

  1. Interesting finding so far, and it appears Carl Zimmer enjoys the work as well.

    Keep up the good work, and I love using this current experiment as an exemplar example of science (independent replication to confirm or refute previous findings)

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  2. NotAnAstrobiologistAugust 2, 2011 at 8:57 PM

    Wow, talk about progress. Well done Rosie and RA who did the sequencing.

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  3. Nice work! One quick question: am I right in thinking that you are comparing cell counts based on acridine orange staining (Wolfe-Simon et al.) to colony counts (your work)? Does acridine orange stain cells that would be incapable of growing enough to form a colony? If that's the case, and Wolfe-Simon's "arsenate-grown" cells are stressed (as she has suggested), then I might expect you to get lower growth yields when you do the arsenate growth experiment.

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  4. @Alexa: Yes, probably yes, and perhaps yes.

    Once I have some data on growth in arsenic I'll probably try some direct counts and also check the OD600, but for now I'm trying to minimize my opportunities to spill 40 mM arsenic all over the lab.

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  5. Seeing those data again now I am wondering if the "added As" condition was growing using the phosphate known to be present (same rate as the "added P" condition) and that the lower amount of biomass formed (not the same as yield - big pet peeve of mine!) was due to As toxicity or simply running out of P (or something else) and stopping?

    As for the nano-SIMS data in the original paper - jury's out - maybe As stuck to macromolecules as per its toxic effect or to the membranes?

    rich

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  6. You will add:

    1. 40 mM Arsenate
    2. 40 mM Arsenate + 3 uM phosphate
    3. 40 mM Arsenate + 1.5 mM phosphate

    Is that correct? Will your 40 mM arseate also contain 3 uM phosphate contamination? If so perhaps a 4th control, just 3 uM phosphate will be useful?

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  7. @Keith: Yes, and also the same three without the arsenate (thus including your 4th control).

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  8. wouldn't be possible to use some kind of radioactive tracing of As ? I'm juste asking.

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