Response to Comments on “A Bacterium That Can Grow Using Arsenic Instead of Phosphorus”

Abstract

Concerns have been raised about our recent study suggesting that arsenic (As) substitutes for phosphorus in major biomolecules of a bacterium that tolerates extreme As concentrations. We welcome the opportunity to better explain our methods and results and to consider alternative interpretations. We maintain that our interpretation of As substitution, based on multiple congruent lines of evidence, is viable. O ur study (1) described the ability of a known microbe, a member of the Gammaproteobacteria (strain GFAJ-1), to survive and grow in the presence of 40 mM arsenate (As) when cultured without deliberately added phosphate (P). We hypothesized that GFAJ-1 incorporates As into biomolecules in place of P, based on evidence ranging from mass spectrometry and x-ray spectroscopy to classical techniques in microbiology. Here, we address specific concerns raised about our procedures and interpretation (2-9).The foundation of our study was a suite of microbial growth experiments demonstrating extreme As tolerance and enhanced growth in +As/-P media as compared with -As/-P controls (1). These experiments have been criticized because of the presence of trace P in the experimental media. Benner (3) suggests that this contamination came as an impurity with the As added to +As/-P experiments and that the level of contamination is unknown. If this were so, the experiments would be fundamentally flawed; however, to clarify the methods described in the Supporting Online Material (SOM) accompanying our original study (1), medium mineral salts were initially made up as -As/-P, without added vitamins or glucose. The P content of the batch of this mixture used in all experiments was 3.7 T 0.8 mM (T2 SD; table S1, 5 April 2010 batch) (1). For every experiment, vitamins, glucose, and either P or As were added to this mixture, as described in "Materials and Methods" in the SOM (1). In the two batches of medium to which we added As along with glucose and vitamins, we measured P contents of 2.9 T 0.6 and 2.7 T 0.6 mM (table S1) (1). These values are statistically indistinguishable from the P content of the -As/-P mineral salt medium. Therefore, the P background presumably came from the mineral salts, not from the added As, and was effectively identical in all treatments (~3 mM).Several comments suggest or imply that this background P was sufficient to sustain growth in the +As/-P experiments (2,3,5,6,8,9). We do not find these arguments compelling given the poor growth in the control experiment compared with the +As/-P experiment, both of which contained the same P background. Any alternative hypothesis must account for this basic observation.Foster (9) hypothesizes that the enhanced growth we observed upon addition of As compared with the -As/-P control can be explained if we inadvertently selected for a microbe that retains a high-affinity P uptake mechanism (Pst) that is stimulated by arsenate. If operational, we would expect evidence of arsenate detoxification processes, such as arsenate reduction or methylation, because all phosph...

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Genome of Halomonas Strain GFAJ-1, a Blueprint for Fame or Business as Usual

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