Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells

Abstract

A strain of Halomonas bacteria, GFAJ-1, has been claimed 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.

Fig. 1. Growth curves of GFAJ-1 in AML60 medium supplemented with different concentrations of phosphate

Each line is the mean of 10 replicate 300 μl cultures in wells of a Bioscreen C Growth Analyzer. The phosphate additions used to replicate the ‘−P’ and ‘+P’ conditions of (1) are indicated.

Fig. 2. Integrity of GFAJ-1 chromosomal DNA after long-term storage

Lanes: 1 and 7: HindIII digest of lambda DNA; 2: H. influenzae chromosomal DNA; 3–6: GFAJ-1 chromosomal DNA grown in the specified combinations of As and P (−As: no arsenate; +As, 40 mM arsenate; −P, 3 μM added phosphate, +P, 1500 μM added phosphate). Panel A. ~100 ng of GFAJ-1 DNA immediately after purification. Panel B. 200 ng of the same DNAs after 2 months storage in Tris EDTA at 4°C. Panel C. The same DNAs as B, but 800 ng/lane and after 10 min at 95 °C.

Fig. 3. LC-MS analysis of arsenate in purified and CsCl fractioned DNA from arsenate-grown GFAJ-1 cells

Representative extracted ion chromatograms for arsenate (mass-to-charge ratio, m/z = 140.9174 +/− 3 ppm) are shown as the chromatographic retention time in minutes plotted against intensity in ion counts. Sample identity is indicated to the right, along the axis extending into the page. DNA from arsenate-grown GFAJ-1 cells (+As/−P undigested gDNA) was analyzed by LC-MS at a 1:10 dilution, as were the water wash (+As/−P wash of gDNA), the same DNA following washing and enzymatic digestion (+As/−P washed, digested DNA), and finally, fractions of the same DNA after a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8, with DNA concentrating in Fractions #6, #7, and #8). Potassium arsenate standards (Std 1.7e-6 to 1.7e-8 [M]) and a water blank were also analyzed. One of four representative experiments is shown.

Fig. 4. LC-MS analysis of deoxynucleotides from purified and CsCl fractioned DNA from arsenate-grown GFAJ-1 cells

Representative extracted ion chromatograms are shown as the chromatographic retention time in minutes plotted against intensity in ion counts. One of four representative experiments is shown. A. and B. Extracted ion chromatograms for A. deoxyadenosine-phosphate (dAMP; m/z = 330.0609 +/− 5 ppm) and B. its arsenate analog deoxyadenosine-arsenate (dAMA; m/z = 374.0087 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). To keep the peak on scale, the signal for +As/−P washed, digested gDNA has been multiplied by 0.5. This observed large peak matches the known retention time of dAMP. C. and D. Extracted ion chromatograms for C. the dideoxynucleotide deoxyadenosine-phosphate (dAMP-dAMP; m/z = 643.1185 +/− 5 ppm) and D. its mono-arsenate analog deoxyadenosine-arsenate-deoxyadenosine-phosphate (dAMA-dAMP; m/z = 687.0663 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). Partially digested −As/+P DNA shows a large peak at the exact mass of dAMP-dAMP.

Fig. 4. LC-MS analysis of deoxynucleotides from purified and CsCl fractioned DNA from arsenate-grown GFAJ-1 cells

Representative extracted ion chromatograms are shown as the chromatographic retention time in minutes plotted against intensity in ion counts. One of four representative experiments is shown. A. and B. Extracted ion chromatograms for A. deoxyadenosine-phosphate (dAMP; m/z = 330.0609 +/− 5 ppm) and B. its arsenate analog deoxyadenosine-arsenate (dAMA; m/z = 374.0087 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). To keep the peak on scale, the signal for +As/−P washed, digested gDNA has been multiplied by 0.5. This observed large peak matches the known retention time of dAMP. C. and D. Extracted ion chromatograms for C. the dideoxynucleotide deoxyadenosine-phosphate (dAMP-dAMP; m/z = 643.1185 +/− 5 ppm) and D. its mono-arsenate analog deoxyadenosine-arsenate-deoxyadenosine-phosphate (dAMA-dAMP; m/z = 687.0663 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). Partially digested −As/+P DNA shows a large peak at the exact mass of dAMP-dAMP.

Fig. 4. LC-MS analysis of deoxynucleotides from purified and CsCl fractioned DNA from arsenate-grown GFAJ-1 cells

Representative extracted ion chromatograms are shown as the chromatographic retention time in minutes plotted against intensity in ion counts. One of four representative experiments is shown. A. and B. Extracted ion chromatograms for A. deoxyadenosine-phosphate (dAMP; m/z = 330.0609 +/− 5 ppm) and B. its arsenate analog deoxyadenosine-arsenate (dAMA; m/z = 374.0087 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). To keep the peak on scale, the signal for +As/−P washed, digested gDNA has been multiplied by 0.5. This observed large peak matches the known retention time of dAMP. C. and D. Extracted ion chromatograms for C. the dideoxynucleotide deoxyadenosine-phosphate (dAMP-dAMP; m/z = 643.1185 +/− 5 ppm) and D. its mono-arsenate analog deoxyadenosine-arsenate-deoxyadenosine-phosphate (dAMA-dAMP; m/z = 687.0663 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). Partially digested −As/+P DNA shows a large peak at the exact mass of dAMP-dAMP.

Fig. 4. LC-MS analysis of deoxynucleotides from purified and CsCl fractioned DNA from arsenate-grown GFAJ-1 cells

Representative extracted ion chromatograms are shown as the chromatographic retention time in minutes plotted against intensity in ion counts. One of four representative experiments is shown. A. and B. Extracted ion chromatograms for A. deoxyadenosine-phosphate (dAMP; m/z = 330.0609 +/− 5 ppm) and B. its arsenate analog deoxyadenosine-arsenate (dAMA; m/z = 374.0087 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). To keep the peak on scale, the signal for +As/−P washed, digested gDNA has been multiplied by 0.5. This observed large peak matches the known retention time of dAMP. C. and D. Extracted ion chromatograms for C. the dideoxynucleotide deoxyadenosine-phosphate (dAMP-dAMP; m/z = 643.1185 +/− 5 ppm) and D. its mono-arsenate analog deoxyadenosine-arsenate-deoxyadenosine-phosphate (dAMA-dAMP; m/z = 687.0663 +/− 5 ppm). DNA from arsenate grown GFAJ-1 cells (+As/−P washed, digested gDNA) was washed, digested, and analyzed by LC-MS, as was the same DNA following a CsCl gradient purification and digestion (+As/−P CsCl Fractions #1 – #8). Partially digested −As/+P DNA shows a large peak at the exact mass of dAMP-dAMP.