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Biological Chemistry

Two Studies Rebut Arsenic-Based Life

Biochemistry: Controversial microbe is likely a phosphorus scavenger

by Carmen Drahl
July 16, 2012 | APPEARED IN VOLUME 90, ISSUE 29

Credit: Science
Scanning electron micrograph of GFAJ-1.
Credit: Science
Scanning electron micrograph of GFAJ-1.

The periodic table of life is holding firm. A year and a half after a study made the controversial claim that a bacterium could weave arsenic into its biomolecules—thereby expanding the chemical definition of life—publications from two independent teams now refute that claim (Science, DOI: 10.1126/­science.1218455; 10.1126/science.1219861).

Credit: Courtesy of Marshall Reaves
Redfield sent these GFAJ-1 samples to her collaborators.
Credit: Courtesy of Marshall Reaves
Redfield sent these GFAJ-1 samples to her collaborators.

The new data indicate that the infamous microbe, known as GFAJ-1, does not incorporate arsenic into its metabolites or DNA when grown in ultrahigh concentrations of arsenic. The work also suggests that contamination could explain some of the conclusions drawn in the original report.

The new results “provide good evidence that GFAJ-1 does not appear to ‘rewrite the textbooks,’ ” says bioorganic chemist Kent S. Gates at the University of Missouri, who wasn’t involved with any of the studies.

A team led by Felisa Wolfe-Simon made a major splash in 2010 after reporting in the journal Science that GFAJ-1, which they’d scooped out of California’s arsenic-rich Mono Lake, survived by using arsenic in place of phosphorus.

The report sparked a vociferous backlash. In 2011, when Science published the paper in print, it was accompanied by eight critiques.

One of the two new studies was conducted by an outspoken critic of the original report, microbiologist Rosemary J. Redfield of the University of British Columbia, along with genomics professor Leonid Kruglyak of Princeton University and coworkers. Using liquid chromatography-mass spectrometry, the team verified that GFAJ-1 DNA had no covalently bound arsenic. Their experiments and conclusions have been public for some time, but this marks the debut of that work in a peer-reviewed journal. Redfield published regular updates and results on her blog and published the manuscript the team submitted to Science to arXiv, a repository for preprints of research papers.

The other study, from microbial physiologists Julia A. Vorholt and Tobias J. Erb and colleagues at ETH Zurich, used inductively coupled plasma mass spectrometry and ultrapure reagents to demonstrate that phosphorus-starved GFAJ-1 scavenges trace amounts of phosphorus from its culture medium to survive.

The new work’s authors “are convinced they have tightly shut the door on arsenic incorporation into biomolecules,” Wolfe-Simon, now at Lawrence Berkeley National Laboratory, wrote in an e-mail. “We respect the Science editors and authors of the current papers but think low amounts of arsenic incorporation may be challenging to find and unstable once cells are opened.”

“We’re not trying to rule out that there’s not a single atom of arsenic anywhere,” Kruglyak says. “The point is that once we’re talking about those types of levels, the bacteria are not using it productively to grow, which was the claim of the original paper.”

In addition to Wolfe-Simon, the original work had 11 other coauthors. Those collaborators reached by C&EN emphasized the need for more study of GFAJ-1.

“What can I say? Chemistry is vindicated,” says Steven A. Benner of the Foundation for Applied Molecular Evolution in Florida, who has disagreed with Wolfe-Simon’s interpretations since her findings were published.“If I were Felisa, I would say, ‘It is clear that our interpretation was wrong’ and move on.”

Reaction To Arsenic-Based Life Rebuttals

Two new studies report that the bacterium GFAJ-1 does not substitute arsenic for phosphorus in its biomolecules, as was previously claimed. Here are select excerpts from researchers’ responses to this news.

Felisa Wolfe-Simon, lead author of original study, Lawrence Berkeley National Laboratory:

“The original GFAJ-1 paper emphasized tolerance to arsenic, but suggested the cells required phosphorus, as seen in these two new papers. However, our data implied that a very small amount of arsenate may be incorporated into cells and biomolecules, helping cells to survive in environments of high arsenate and very low phosphate. Such low amounts of arsenic incorporation may be challenging to find and unstable once cells are opened.

So key questions are, how do these cells thrive in lethal concentrations of arsenic? And where does the arsenic go? Answers to these questions merit the most thorough and careful analysis we can achieve.

We expect to prepare a paper in the next few months. As an additional point, these new Science papers support strongly held wisdom that arsenic CANNOT substitute for phosphorus in biomolecules.

These authors have made it clear that they are convinced they have tightly shut the door on arsenic incorporation into biomolecules. We respect the Science editors and authors of the current papers, but think low amounts of arsenic incorporation may be challenging to find and unstable once cells are opened so may merit comprehensive and systematic efforts with other assays and conditions.”

Rosemary Redfield, University of British Columbia, corresponding author of a rebuttal:

“Science (the process, not the journal) is ‘self-correcting’ in the sense that individual scientists work to correct the errors made by other scientists. Researchers who have published erroneous work sometimes say that they are happy that their work has stimulated further research, when they should be apologizing for having created the circumstances that necessitated the additional work.”

Michael H. New, astrobiology discipline scientist in NASA’s Planetary Science Division at NASA headquarters:

“NASA supports robust and continuous peer review of any scientific finding, especially discoveries with wide-ranging implications. It was expected that the 2010 Wolfe-Simon et al. Science paper would not be exempt from such standard scientific practices, and in fact, was anticipated to generate significant scientific attention given the surprising results in that paper. The two new papers published in Science on the micro-organism GFAJ-1 exemplify this process and provide important new insights. Though these new papers challenge some of the conclusions of the original paper, neither paper invalidates the 2010 observations of a remarkable micro-organism that can survive in a highly phosphate-poor and arsenic-rich environment toxic to many other micro-organisms. What has emerged from these three papers is an as yet incomplete picture of GFAJ-1 that clearly calls for additional research.”

Ariel Anbar, senior coauthor of the original study, at Arizona State University (reached while traveling):

“I am glad that there are new data to work with. That’s what is needed to figure things out. That’s how science works.”

Ronald S. Oremland, senior coauthor of original study, at the U.S. Geological Survey:

“While the progress of science is usually thought to move forward in a straightforward, logical fashion, in reality it often progresses more like that of a pin-ball in an arcade game. While the results of Reaves et al. (2012) and Erb et al. (2012) may eventually firmly close the door on the validity of the original arsenic-life hypothesis, at this point I would say it is still just a tad ajar, with points worthy of further study before either slamming it shut or opening it further and allowing more light inside.

The lab I run is re-examining the original experiments after devising a new medium that eliminates the problem of background P contained in the reagents first employed.

One reasonable possibility for the disparity in this research is that this trait of arsenate-dependent growth has been lost over time. So in addition to conducting further investigations on the arsenate-enhancement of growth at low phosphorus on the currently cultivated strain, efforts should be undertaken once again to isolate new strains from Mono Lake using the reconfigured zero background phosphorus medium that our USGS lab has begun using.”

Samuel M. Webb, coauthor of original study, beam line scientist at the Stanford Synchrotron Radiation Lightsource:

“My role in the original study was that I worked with Felisa to do the X-ray absorption studies of whole cells of GFAJ-1. I was impressed that the As content was significant and that we were able to get spectra from groups of whole cells. The X-ray absorption techniques are best for attempting to identify the speciation of chemical form of the element present, in this case, of course, the As. What struck me was that As was not apparently just present as As(V) in cells/sorbed matter, and had not been reduced or bound to S-thiols or As-S precipitates as many As-tolerant bacteria/plants/yeast often do. The As speciation could not be conclusively determined (its local structure determination only), but was consistent with As present in any number of As-biomolecules.

I certainly still believe in our XAS results–those were done on whole cells that had been kept intact and show that there is something interesting and I believe novel occurring. The new papers certainly bring interesting observations to the table that do not support some of the earlier, original hypotheses that were made. These observations were made using a different battery of sophisticated tests on various extracts of cells. Overall, these are great ways to expand the knowledge about GFAJ-1 and narrow down how it manages both As and P in its biochemistry. I think only through more research can we find out what this organism is truly doing, and how.

I haven’t participated in the laboratory experiments that are continuing to be performed by many of the members of the original paper, as I am not primarily a microbiologist. However, we do have an active proposal to do further XAS measurements at SSRL and SLAC when the samples are ready. As synchrotron time is often scarce and hard to get, it takes extensive planning to gather the materials and samples needed, especially since there are many directions in which the current research is progressing.

I think the focus should be less on controversy and such and more about determining what the organism is doing. Regardless if there is As in DNA or not, and to what end the original hypothesis comes to, the fact is still that GFAJ-1 can tolerate extremely high As content and thrive there under low P conditions, and I think there is still interesting biochemistry occurring which is worthy of further study.”

Thomas R. Kulp, coauthor of original study, now at the State University of New York, Binghamton:

“I stand by what we observed in the laboratory and reported in the original paper regarding the apparent stimulatory effect of As on the growth of GFAJ-1 at limiting P concentrations. Both Reaves et al. and Erb et al. suggest that low levels (~3 µM) of phosphate contamination in our media were sufficient to sustain the growth that we attributed to substitution of As for P in biomolecules. This is supported by their observations of modest growth at similar phosphate concentrations without additional As. In our original experiments both the As-amended and non-As conditions contained equal amounts of P contaminant (~3 µM), but we observed significant growth at this P concentration only in the presence of added As. Reaves et al. were unable to achieve growth of GFAJ-1 under any conditions without additional organic amendments (glutamate). By contrast, Erb et al. were able to grow the organism using our described media preparation without the need for additional organic amendments, although they did not observe the same stimulatory effect for As. The cause for the discrepancy between our growth results is not known. Before we “throw away the baby with the bath water,” I think that it bears further investigation to determine the cause of the apparent stimulatory effect of As in our original cultures.

Both of these new studies also utilized various chromatographic mass spectroscopy methods to rule out the presence of As in nucleic acids or nucleotides, a significant but very over-hyped claim of our original paper. Our interpretations were based on several lines of evidence including radio-tracer assays and X-ray spectroscopy. However, if superior methods cast doubt on this conclusion, it still begs the question of how GFAJ-1 was able to utilize As to sustain growth at limiting P concentrations in our growth experiments. As Erb et al. conclude in their report, GFAJ-1 has proven to be a very interesting bacterium and much remains to be learned about how this organism is able to tolerate arsenate:phosphate ratios that are orders of magnitude higher than those previously reported for other As-tolerant species.

I have recently left the USGS to start a new research program and lab at Binghamton University. For the past year I have been working to get these new facilities set up and operational. Given the fact that the current GFAJ-1 strain has been carried for well over a year through many transfers, a good plan might be to start from scratch and attempt to isolate this (or a similar organism) again directly from the Mono Lake sediment and attempt to replicate our As-dependent growth results with a new isolate. I would encourage other researchers to attempt the same.”

Kent. S. Gates, University of Missouri:

“Although many have pointed out bumps and glitches in the history of the Wolfe-Simon paper, at the end of the day, the scientific process seems to have worked pretty well here. The Wolfe-Simon team made claims that certainly captured the imagination of both scientists and the public. The claims deserved to be tested and a number of scientists stepped up to do the job. It is important that Oremland and Blum provided the GFAJ-1 strain so that others could carry out their own experimental tests of the findings reported in the original Science paper. The new results of both Erb et al. and Reaves et al. provide good evidence that the GFAJ-1 does NOT appear to “rewrite the textbooks” by executing a wholesale replacement of arsenate for phosphate in its macromolecules and metabolome. The work of Erb et al. confirms that GFAJ-1 is remarkably resistant to arsenate and any future studies of the organism can now properly focus on this interesting property.”

David Sanders, Purdue University:

It’s not enough to say science is self-correcting, contends David Sanders, who studies enzymatic phosphoryl transfer at Purdue University and has written critically about the original work in the blog Retraction Watch. “The text in the original paper misrepresents data in the supplementary information,” and so the paper should be retracted by Science, he says.

Steven A. Benner, Foundation for Applied Molecular Evolution:

“What can I say? Chemistry is vindicated,” says Steven A. Benner of the Foundation for Applied Molecular Evolution in Florida, who has disagreed with Wolfe-Simon’s interpretations since her findings were published. Both of the new refutations are studies of extremely high quality, Benner says. “I cannot imagine that any set of experiments will ever lead to the same conclusions that Felisa drew in her Science paper 18 months ago.

“If I were Felisa, I would say, ‘It is clear that our interpretation was wrong’ and move on.”


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