PRIORITY MAIL [+]Enlarge Credit: Courtesy of Marshall Reaves

Just after Thanksgiving, Marshall L. Reaves got a package in the mail. He’d been anticipating it for months. And scientists worldwide were anticipating the answers that package might bring.

GROWING AT LAST [+]Enlarge Credit: Courtesy of Rosie Redfield CLEANUP TIME [+]Enlarge Credit: Courtesy of Marshall Reaves AT ISSUE [+]Enlarge SOURCE OF CONTROVERSY [+]Enlarge Credit: Henry Bortman

This month, Redfield posted online mass spectrometry data Reaves obtained by analyzing the package’s contents along with the conclusion she and her colleagues reached based in part on those results—that the DNA from GFAJ-1 contains no arsenic. Compared with the high-profile press conference, front-page headlines, and scientific backlash the original report generated, which included eight rebuttals published in Science (C&EN, June 6, 2011, page 7), the response to Reaves’s preliminary data has thus far been sedate. But the so-called arsenic-life paper has left a noticeable footprint on science and science communication just over a year after its publication.

The lead researcher on the original report, Felisa Wolfe-Simon, has been delving deeper into GFAJ-1’s biology and chemistry. Other researchers are also studying GFAJ-1, which stands for “Give Felisa a Job,” a name designed to highlight Wolfe-Simon’s quest to trade temporary scientific positions for a permanent post. Still other scientists are looking into arsenic biochemistry in general.

Meanwhile, Redfield, who goes by Rosie, has been keeping scientists and journalists alike engaged with updates on her efforts to replicate Wolfe-Simon’s work, all posted to her blog RRResearch. The mainstream press continues to cover the unfolding story and news and commentary related to the saga abound on the social web, often labeled with the Twitter hashtag #arseniclife.

If arsenic-based DNA really existed, it would fundamentally alter scientists’ understanding of the chemistry of life on Earth and point to chemistry that, at least in theory, could be used to sustain life elsewhere in the universe. For that claim to be right, however, 50 to 100 years of chemical precedents about arsenic toxicity and arsenic biomolecule stability would have to be wrong, says chemist Steven A. Benner of the Foundation for Applied Molecular Evolution in Florida, who has questioned Wolfe-Simon’s findings since their publication. “No chemist is going to let that go down with only the experiments that appeared in the original paper,” he says.

Redfield wasted no time putting the claims to the test. To some extent she thinks it’s a waste of scientific time to try to replicate the work. But “given that the work had that big a splash, it should be tested, not just discarded,” she says. And as a longtime practitioner of open science, she also saw in GFAJ-1 an opportunity to showcase openness in research “under circumstances where people would be really excited to see the results.”

Redfield’s blog chronicles how at first she had a tough time getting GFAJ-1 to grow reproducibly in media that is arsenic-rich but is low in phosphorus, the key conditions in the Science paper. In her blog’s comments section others left suggestions for experiments and controls. Last November, the bacteria finally started growing consistently. Redfield isolated DNA from GFAJ-1 grown in arsenic-rich medium as well as in several types of control media. Then she shipped everything off to Princeton, where Reaves, Rabinowitz, and genomics professor Leonid Kruglyak were waiting.

Reaves further purified Redfield’s GFAJ-1 DNA samples with cesium chloride density-gradient centrifugation, which separates DNA from impurities based of density. “It can remove a lot of other things that might want to stick to DNA because of charge,” he explains. Then he removed excess salts left over from the cesium chloride step and used a pair of enzymes to chew up the DNA to its individual nucleotide building blocks. After one last cleanup to remove any residual protein, he examined the nucleotides with liquid chromatography-mass spectrometry (LC/MS), which physically separates nucleotides on the basis of polarity and then analyzes their mass.

None of the purified DNA samples showed any signs of containing an arsenic-containing DNA building block, Reaves says. He looked for masses that would be indicative of individual arsenic deoxynucleotides, as well as for free arsenate that might result from arsenic-based DNA falling apart in water. Work by other labs suggests that DNA with weird nucleotides might not get completely chewed up by normal enzymes (Nat. Chem. Biol., DOI: 10.1038/nchembio.2007.39; Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1017261108). So he also searched for pairs or small stretches of nucleotides that might occur if that were the case. He came up empty time and again.

Reaves also examined samples of DNA from GFAJ-1 that had been grown in arsenic-rich media, but without doing any of the purification steps. That’s when he detected arsenate. “It’s kind of stuck to the DNA” noncovalently, he says, but he can remove most of it by washing the DNA with water.

Given the limited amount of data presented so far, experts in nucleic acid MS are cautious about judging the Redfield-Princeton team’s results. But they are confident the study’s methods are sound, and one expert told C&EN he is reasonably in agreement with the study’s conclusions.

“Most scientists hedge their bets until all the data is presented,” says Patrick A. Limbach of the University of Cincinnati, an expert in nucleic acid MS. On the basis of what he’s read, he thinks the team’s LC/MS approach is a solid way to check for arsenic incorporation into individual DNA building blocks. “It would be speculation to extrapolate these few data pieces towards the entire picture,” but given the caveat of limited data “I would be comfortable with their conclusion,” he says. “Their data strongly suggests that it is unlikely that arsenic is being integrated into the genomic DNA of the sample this group isolated, purified, and analyzed.”

The Princeton team’s process for preparing the DNA for LC/MS analysis “is exactly what I would’ve tried,” says Michael G. Bartlett, whose work at the University of Georgia Center for Drug Discovery includes MS of oligonucleotides. But he adds it’s tough for him to evaluate the team’s conclusions based on what’s been posted online. He recommends a complementary experiment—performing ion chromatography of the intact DNA, without chopping it up, followed by inductively coupled plasma MS. In that method, “you would combust the entire DNA and test for arsenic,” he says. The plasma method tends to produce a less noisy baseline than LC/MS, so vanishingly small amounts of arsenic species would be more easily detectable if they are present. Defenders of the arsenic-based-life claim might argue that using an enzyme degradation method with arsenic-based DNA would affect the experiment’s outcome, Bartlett notes. The plasma method, he says, would make enzyme degradation unnecessary.

Proponents of arsenic-based life might also contend that Redfield tweaked Wolfe-Simon’s GFAJ-1 growth conditions—Redfield added a tiny amount of phosphorus, an amino acid, and a source of potassium. It’s not clear how much phosphorus was present in Wolfe-Simon’s original conditions, so proponents could contend that Redfield’s GFAJ-1 had enough phosphorus to survive and so wouldn’t bother to incorporate arsenic.

But Kruglyak doesn’t think those adjustments invalidate their study. Redfield added only enough phosphorus to match levels of contamination she estimated from the original report, and potassium is abundant in Mono Lake and present in a different form in some of Wolfe-Simon’s bacterial growth media. “It’s always something one can claim, that nobody’s reproduced conditions exactly,” Kruglyak says. The only real response to that, he says, would be to request samples from Wolfe-Simon and her collaborators and test those.

The Princeton-Redfield team plans to replicate their results and submit them to Science for peer review. “We thought that the record should be tested and corrected if needed,” Kruglyak says. “And we thought it would be kind of fun to do it ourselves instead of waiting around for somebody else to do it.”

The Redfield-Princeton team’s work comes on the heels of the first genome sequence for GFAJ-1. Last December, a multi-institution team deposited the draft sequence into GenBank, the freely available National Institutes of Health genetic-sequence database. Leading the effort was University of Illinois, Chicago, microbiologist Simon Silver, who early in 2011 published a scathing critique of the arsenic-based-life work (FEMS Microbiol. Lett., DOI: 10.1111/j.1574-6968.2010.02202.x). Silver says that like Redfield, his team had a hard time getting GFAJ-1 to grow consistently. Once his team determined the genome sequence, though, he was surprised by how few of GFAJ-1’s genes were known to be involved with arsenate resistance, given that the microbe managed to eke out an existence in an environment teeming with normally toxic arsenic. Even the workhorse lab bacterium Escherichia coli has more arsenate-resistance genes, he says.

The genome alone doesn’t answer the question of whether arsenic replaces phosphorus in the backbones of GFAJ-1’s nucleic acids, Silver says. He’s not sure that any results would convince Wolfe-Simon and her coauthors to change their minds about that. However, the genome might provide clues about how the microbe copes with an abundance of an element that should be lethal. His team is planning to see how GFAJ-1’s genome compares with genomes of three other microbes that grow in arsenic-rich environments.

Ronald S. Oremland, a coauthor of the controversial Science paper and Wolfe-Simon’s postdoctoral adviser at the time, declined to comment on Reaves’s results. “That is not a reflection on my views concerning the validity of the work,” he says. “I am old-school in following the scientific publication review process, reserving commentary for later.” His lab isn’t working on GFAJ-1 right now, but they have cultures of the microbe and may do more work in the future. He did not renew Wolfe-Simon’s postdoctoral appointment but says he helped her make arrangements to find a new home base for her arsenic research.

Oremland, a senior scientist with the U.S. Geological Survey, says he’s received plenty of criticism about the GFAJ-1 work, some of it constructive but much of it venomous. “My fear is that scientists will be afraid to test radical ideas” because of the arsenic-based-life saga, he says. “If you don’t think, and you don’t try, and you don’t test, then everyone just keeps doing the same incremental stuff.” The Science paper’s conclusions accurately reflect what the data looked like to him and his coauthors at the time, he says. “We put this idea out there so it could be tested.”

“Even if we are dead wrong with this arsenic-DNA business, with a bit more work this bug could shed light on the limits of what microbes can and can’t do.”

Asked to comment on whether he is open to changing his mind about whether GFAJ-1 has arsenic DNA, Oremland said, “I’m not going to go down with the ship.” If other groups’ work makes it clear that he and his coauthors overreached, “I’ll say I was wrong. I make mistakes. That’s the way science works,” he says. “But you might find Felisa holding on a bit tighter.”

“We are thrilled that our results are stimulating more experiments from others as well as ourselves,” Wolfe-Simon wrote in an e-mail. She is now a National Aeronautics & Space Administration Astrobiology Research Fellow in structural biologist John A. Tainer’s lab at Lawrence Berkeley National Laboratory. “We do not expect to see arsenate in cesium chloride gradient experiments—although this is a great thing to check.” Once GFAJ-1 cells are cracked open, any arsenic linkages in DNA are liable to fall apart, she writes. As a result, “arsenate-containing bands may then also be shifted in the gradient—so that the expected amounts associated with DNA would be undetected because the band is so faint.”


Redfield and coworkers posted GFAJ-1 data to Redfield's blog. Visit these links for select data and explanations. Gel photo: rrresearch.fieldofscience.com/2012/ 01/heres-gel-photo.html CsCl MS data: rrresearch.fieldofscience.com/2012/ 01/csclmass-spectrometry-data.html More power of CsCl gradients: rrresearch.fieldofscience.com/2012/ 01/more-power-of-cscl-gradients.html Control results: rrresearch.fieldofscience.com/2011/ 12/some-control-results-dont-get-excited.html

“That concern doesn’t make much sense,” Redfield says. She performed a gel analysis to ensure that GFAJ-1 DNA from cells grown in arsenate-rich conditions was not falling apart prior to Reaves’s purification. She posted those results to her blog on Jan. 14. The gel “shows that the DNA from arsenate-grown cells is no more degraded than DNA from phosphate-grown cells, both immediately after isolation and after two months’ storage at 4 °C,” Redfield says. Combined with the LC/MS data, Redfield’s evidence gives arsenic-based DNA “no place to hide,” Reaves adds.

In Tainer’s lab, Wolfe-Simon is trying multiple tactics to learn more about what happens to arsenic inside GFAJ-1. “We are focused on isolating and characterizing the ribosome via X-ray crystallography. Additionally, we are looking at the DNA/RNA and other cellular metabolites with quadrupole time-of-flight LC/MS,” she says. “We will be purifying DNA, RNA, and proteins and seeing whether or not the arsenic tracks with these components.”

In a poster presented at last December’s American Geophysical Union meeting, Wolfe-Simon reported progress on optimizing GFAJ-1’s growth with a combination of carbon sources and amino acids, as well as progress in preparing samples for nucleic acid stability testing, ribosome crystallization, and MS analyses. “We think structure is the way to go,” Wolfe-Simon says. “We want to solve the structure of the ribosome and see what it’s going to tell us,” she adds. “Is the arsenic there? Is it randomly distributed? Is it in specific places?” she says. “We’re going to look at the RNA to see if [arsenic] is in the backbone.”

“We’re super excited about our next experiments,” she says. “What’s nice about science is the truth will reveal itself.”

To some researchers, however, an attempt to crystallize GFAJ-1’s ribosome to check whether it contains arsenic is like leaving the low-hanging fruit behind and clambering straight for the treetops. The focus at first should be on more straightforward tests that get at the heart of the is-there-or-isn’t-there-arsenic-in-the-DNA question, says Benner. He’d like to see Wolfe-Simon grow GFAJ-1 in the presence of radioactive arsenate and then perform multiple fractionations—separations of the various cellular components—while tracking the radioactivity to see where the arsenate goes. In the original report, “she did one fractionation. She started down the road a chemist understands,” Benner says. But she didn’t do enough rounds of fractionation to see whether radiolabeled arsenate could end up in specific molecules, such as DNA, he adds.

“Crystals of bacterial ribosomes have the resolution to yield atomic models these days,” says Venkatraman Ramakrishnan of the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, who shared the 2009 Nobel Prize in Chemistry for ribosome crystallization work. But it’s not necessary to crystallize GFAJ-1’s ribosome to put the arsenic-based-life controversy to rest, he says. “Crystallization of ribosomes from a new species is a nontrivial undertaking that can take many years, and possibly never succeed,” he says. He suggests that the team focus on isolating ribosomal RNA, or nucleotides from ribosomal RNA, and determining, with radioactivity or other tools, whether arsenic ends up there.

Even if GFAJ-1 turns out to be a microbe that can tolerate arsenic while scavenging what phosphorus it can from phosphorus-poor surroundings, that would still be fascinating, Ramakrishnan adds, “although certainly not as dramatic as incorporating arsenic for phosphorus in its nucleic acids.”

Other labs besides Redfield’s and Silver’s now have access to GFAJ-1. Oremland did not disclose to whom he’s sent samples. Oremland adds that he doesn’t have an accurate count of how many samples he’s given out but says that the cultures are available from collections in the U.S. and Germany.

Even as follow-up on Wolfe-Simon’s report continues, the work has spurred studies of arsenate chemistry and biochemistry in other labs. At Harvard Medical School, A. Michael Sismour is trying to make arsenate dinucleotides—the chemical motif that would appear in arsenic DNA, if it were to exist. Sismour, who earned his Ph.D. with Benner and is now a postdoctoral fellow in George M. Church’s lab, says that after the arsenic-based-life paper came out he took a look at the literature on arsenic biomolecules, which largely dates to the 1960s through the 1980s, and says he found small contradictions when it came to how long the molecules could last. He figured modern techniques and instrumentation might provide new insights, so he decided to examine the matter as a side project.

So far, Sismour has made arsenate dinucleotides joined by a 5ʹ-5ʹ linkage, which does not occur in standard DNA biochemistry. Making arsenate dinucleotides with the 5ʹ-3ʹ linkage that occurs in DNA has proven challenging. If he does manage to get there, he says he’ll determine the compounds’ half-lives in water.

Chemical precedent suggests that the type of arsenic diesters that would exist in arsenic DNA fall apart in a fraction of a second under biological conditions (ACS Chem. Biol., DOI: 10.1021/cb2000023). But Benner says that the arsenic-life paper at least lets scientists entertain the possibility that in a very cold environment where reaction rates slow down, perhaps on a distant planet somewhere, arsenic-containing DNA’s liability of falling apart easily might be less of a problem. “If we understand what the hydrolysis rates are,” Sismour says, “we might be able to understand what temperature ranges might allow arsenic life to exist.”

Half a world away in Israel, Weizmann Institute of Science biochemist Dan S. Tawfik is thinking about how enzymes that have evolved to work with phosphate might deal with arsenate. In response to the original GFAJ-1 report, Tawfik teamed with Ronald E. Viola of the University of Toledo to publish a summary of research efforts in that area (Biochemistry, DOI: 10.1021/bi200002a).

To survive, the GFAJ-1 microbe had to come up with some way of coping with high concentrations of arsenate, whether or not arsenic is incorporated into GFAJ-1’s DNA, Tawfik reasons. Since writing the summary, his team has set out to learn about arsenic-survival measures that organisms could possibly have developed in arsenic-rich environments.

Work by Viola and others suggests many enzymes that bind to or use phosphate have a hard time telling the difference between phosphate and arsenate (Inorg. Chem., DOI: 10.1021/ic981082j; Acta. Cryst., DOI: 10.1107/S0907444904026411; J. Biol. Chem.1981, 256, 5981). That lack of discrimination could be lethal with high concentrations of arsenate present. Tawfik is focusing on a bacterial transport protein that he says binds phosphate one thousand times more strongly than arsenate. In preliminary work, his team used X-ray crystallography to obtain molecular-level close-ups of the protein bound to both arsenate and phosphate. It’s not uncommon for different substrate analogs to bind to a protein with a highly different affinity than the natural substrate. But it may be, Tawfik speculates, that at some point on the primitive Earth, some proteins had to evolve to distinguish between phosphorus and arsenic, an adaptation that would rarely be necessary today.

Few scientists believe GFAJ-1 has arsenic-containing DNA, Tawfik says. “But that doesn’t mean there won’t be other intriguing findings related to this microbe” or related to arsenic biochemistry.