Absence of Detectable Arsenate in DNA from Arsenate-Grown GFAJ-1 Cells

Abstract

A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.Wolfe-Simon et al. isolated strain GFAJ-1 from the arsenic-rich sediments of California's Mono Lake by its ability to grow through multiple subculturings in artificial Mono Lake medium AML60 that lacked added phosphate but had high concentrations of arsenate (+As/ −P condition) (1). Because GFAJ-1 grew in −P medium only when arsenate was provided, and because significant amounts of arsenate were detected in subcellular fractions, growth was attributed to the use of arsenate in place of phosphate. However, the basal level of phosphate contaminating the −P medium was reported to be 3-4 M (1), which previous studies of low-phosphate microbial communities suggest is sufficient to support moderate growth (2). GFAJ-1 grew well on medium supplemented with ample phosphate but no arsenate (1500 M PO 4 , +P/-As condition), indicating that GFAJ-1 is not obligately arsenate-dependent. Wolfe-Simon et al.(1) further reported that arsenic was incorporated into the DNA backbone of GFAJ-1 in place of phosphorus, with an estimated 4% replacement of P by As based on the As:P ratio measured in agarose gel slices containing DNA samples. This † Corresponding author: redfield@zoology.ubc.ca. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript finding was surprising because arsenate is predicted to reduce rapidly to arsenite in physiological conditions (3, 4), and because arsenate esters in aqueous solution are known to be rapidly hydrolyzed (5). We have now tested this report by culturing GFAJ-1 cells supplied by the authors (1) and by analyzing highly purified DNA from phosphate-limited cells grown with and without arsenate.Wolfe-Simon et al. reported that GFAJ-1 cells grew very slowly in AML60 medium (doubling time ~12 hours), and that, when phosphate was not added to the medium, cells failed to grow unless arsenate (40 mM) was provided (1). However, although we obtained strain GFAJ-1 from these authors, in our hands GFAJ-1 was unable to grow at all in AML60 medium containing the specified trace elements and vitamins, even with 1500 M sodium phosphate added as specified in (1). We confirmed the strain's identity using RT-PCR and sequencing of 16S rRNA, using primers specified by Wolfe-Simon et al.(1); this gave a sequence identical to that reported for strain GFAJ-1. We then found that addition of small amounts of yeast extract, tryptone or individual amino acids to basal AML60 medium allowed growth, with do...

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“…The scientific response to the apparent finding of bacteria (GFAJ‐1) living in an arsenic‐rich, phosphate‐free environment,6 and suggesting the existence of arseno‐DNA, was comprehensive: chemists noted that the rates of hydrolysis50 and reduction51 of arsenate esters would predict a prohibitively low stability for arseno‐DNA; furthermore, biologists showed that trace levels of phosphate were present and sufficient for bacteria,7 and found no detectable arsenic in their DNA 52. It transpired that the bacteria are able to tolerate high arsenate/low phosphate environments because they can be extremely selective in their uptake of the two: a phosphate‐binding protein (PBP) up‐regulated by GFAJ‐1 in phosphate‐poor conditions has a 4500‐fold preference for phosphate over arsenate.…”

Contextual sensitivity in scientific reproducibility

“…Unsuccessful attempts to replicate findings in genetics (1), pharmacology (2), oncology (3), biology (4), and economics (5) have given credence to previous speculation that most published research findings are false (6). Indeed, since the launch of the clinicaltrials.gov registry in 2000, which forced researchers to preregister their methods and outcome measures, the percentage of large heart-disease clinical trials reporting significant positive results plummeted from 57% to a mere 8% (7).…”

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“…In July 2012, Science published an article in which Redfield and collaborators replicated the original arsenic study (10). Blogging during the research process was no obstacle.…”