ISSN 19950829, Inland Water Biology, 2011, Vol. 4, No. 3, pp. 400–401. © Pleiades Publishing, Ltd., 2011. Original Russian Text © N.V. Shadrin, 2011, published in Biologiya Vnutrennikh Vod, No. 3, 2011, pp. 92–93.

REVIEWS

On the Article “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus” by F. WolfeSimon, J.S. Blum, T.R. Kulp, G.W. Gordon, S.E. Hoeft, J. PettRidge, J.F. Stolz, S.M. Webb, P.K. Weber, P.C. W. Davies, A.D. Anbar, and R.S. Oremland1 N. V. Shadrin Kovalevskii Institute of Biology of Southern Seas, National Academy of Sciences of Ukraine, ul. Nakhimova 2, Sevastopol, 99011 Ukraine email: [email protected] DOI: 10.1134/S199508291103014X 1

The components of living cells of all organisms are built on the basis of six biogenic elements: carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphor. Phosphor is included in the construction material of nucleotides that are bricks of DNA, RNA, and ATP. A bacterium has been discovered that can use arsenic instead of phosphorus in its constructive metabolism. This unusual bacterium has been isolated from the hypersaline Mono Lake (United States) [12]. It has become a tradition to discover unlikely bacteria and archaea in extreme biotopes. Only hypersaline water bodies contain organisms–archaea with quadratic, rectangular, and rhombic cells [8] and different unique metabolic properties [9]. The properties of Mono Lake create the conditions for unexpected organisms such as this bacterium [11]. The internaldrainage Mono Lake (37°59′39′′N, 119°00′14′′W) is located at a height of 1945 m above sea level in a volcano caldera that formed ~33000 years ago. The area of the lake is 150 km2, the volume is 2.97 km3, and the average depth is 43 m. The salinity of the lake has been growing in recent decades (up to 90–100‰). The lake is alka line, the pH averages 9.8, and the alkalinity (the quan tity of dissolved organic carbon) is 4.3 g/l. The biodi versity in the lake is low enough, but the lake is highly productive. The concentrations of separate elements in hyper saline lakes of volcanic and tectonic origin often exceed those in “regular” water bodies by several orders of magnitude. The highest concentrations of arsenic (15–29 mg/l or 200 μmol/l), which are 2⎯3 orders of magnitude higher than in “normal” water bodies, are noted in the Mono Lake [6, 11]. However, in another hypersalinesoda lake (Searls Lake in the western United States, where the salinity is >300‰), the concentration of arsenic is 3000 μmol/l [6]. Meanwhile, the maximum permissible concentra 1 Sciencexpress

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tion of arsenic in drinking water is 0.13 μmol/l [6]; therefore, it is not surprising that the diversity of organisms using arsenic in their energy metabolism is relatively large just in these lakes [1, 5, 7]. Arsenic, which is close to phosphorus in chemical properties, is the 20th most frequently occurring ele ment in the earth’s crust (0.0005%); however, it was much more abundant in the earth’s crust at the dawn of life [6]. Many different prokaryotes are known that use the oxidation of arsenic to generate energy either by oxidizing arsenite or through arsenate respiration [1, 5, 7]. Anoxygenic photosynthesis with the use of arsenic compounds has also been discovered [4]. However, this was the use of arsenic compounds by prokaryotes in the energy metabolism. The article under review showed the possibility for bacteria to use arsenic in the constructive metabolism as an alterna tive to phosphorus [12]. This is a principally new understanding of the biochemistry of life that affects established ideas which have never been contested before. The content of the article and conclusions are 3– considered below. Phosphate ion ( PO 4 ) behaves 3–

chemically like arsenate ( AsO 4 ) within the limits of biologically important values of pH and Eh [10], which is what determines the high toxicity of arsen ates; cells cannot distinguish them. However, arsen ates are hydrolyzed much more easily and are less sta ble; therefore, they were thought to be incapable of substituting phosphates in the structural substances of a cell. The hypothesis that organisms capable of replacing phosphorus by arsenic in their constructive metabolism can exist in biotopes rich with arsenic was advanced earlier [13]. Having taken a probe of bottom sediments from the Mono Lake as a subculture, the authors of the article placed it in an artificial aerobic 9.8 pH medium containing 10 mmol of glucose, vita mins, and microelements, but no phosphates or any additional organic substances. The passage of the cul

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ture was performed while increasing the concentration 3– of AsO 4 from 100 μmol to 5 mmol. After six passages at an arsenate concentration of 5 mmol, a certain growth of colonies was observed. The GFAJ1 bacte rium strain isolated from the growing colonies was identified to relate to the Halomonadaceae and Gam maproteobacteria family using 16S rRNA sequencing. Representatives of this family can accumulate high concentrations of arsenic in their cells [1, 7]. The iso lated GFAJ1 strain was subsequently kept anaerobi 3– cally on the medium with 40 mmol of AsO 4 and 10 mmol of glucose without phosphorus, and there was a specific growth rate (μmax) of 0.53/days; the number of cells increased by a factor of 20 after 6 days. During incubation on the medium with phosphorus with μmax = 0.86/days and on the medium without phosphorus and arsenic, growth was not observed at all. Research using versatile modern methods showed that arsenic was present in all the most important components of a cell. On the medium with phospho rus (without arsenic), the quantity of phosphorus in cells was 0.54% of dry weight; that of arsenic was 0.001%. On the medium with arsenic (without phos phorus), the quantity of phosphorus and arsenic was 0.019 and 0.19%, respectively. Labeling with radioac 3– tive 73 AsO 4 showed that arsenic was included in all the most important molecules of a cell in which phos phorus was an essential element. The cells that grew on arsenate and phosphorus had a volume of 2.5 ± 0.4 μm3 and 1.5 ± 0.5 μm3. As the research using a transmission electronic microscope showed, the vacuolelike areas were formed during cultivation on arsenate. The authors of the article relate this to the stabilization of an unstable arsenate ion. The ability to use arsenic instead of phosphorus in constructive metabolism is likely a Precambrian atavism (a relict type of metabo lism). GFAJ1 bacterium that can substitute stable phosphate by unstable arsenate in its constructive metabolism was isolated just from the hypersaline water body. The case in point is that the hypersaline conditions relate to biotopes with the lowest activity of water [3]; consequently, the molecules containing 3– AsO 4 in their composition are more stable in such conditions than in freshwater or sea water. Under the conditions of the underice ocean in Jupiter’s moon Europa [2], organisms that use arsenic constructively instead of phosphorus would likely have advantages over those using phosphorus.

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Many questions raised by this discovery have not been answered, but the data presented in this article indicate that an organism with a unique property has been discovered. The biochemical basis for life turns out to be wider than we are used to thinking. This dis covery shows that research on extreme water bodies can offer a lot not only for hydrobiology, but also for biology as a whole. REFERENCES 1. Fisher, J.C., Wallschläger, D., PlanerFriedrich, B., and Hollibaugh, J.T., A New Role for Sulfur in Arsenic Cycling, Environ. Sci. Technol., 2008, vol. 42, no. 1, pp. 81–85. 2. Gaidos, E.J., Nealson, K.H., and Kirschvink, J.L., Life in IceCovered Oceans, Science, 1999, vol. 284, pp. 1631–1632. 3. Grant, W.D., Life at Low Water Activity, Phil. Trans. R. Soc., L. B., 2004, vol. 359, pp. 1249–1267. 4. Hoeft, S.E., Kulp, T.R., Han, S., Coupled Arsenotro phy in a Hot Spring Photosynthetic Biofilm at Mono Lake, California, Appl. Environ. Microbiol., 2010, vol. 76, no. 14, pp. 4633–4639. 5. Lloid, J.R. and Oremland, R.S., Microbial Transfor mation of Arsenic in Environment from Soda Lakes to Aquifers, Elements, 2006, vol. 2, pp. 85–90. 6. Oremland, R.S., Saltikov, C.W., WolfeSimon, F., and Stolz, J.F., Arsenic in the Evolution of Earth and Extra terrestrial Ecosystems, Geomicrobiol. J., 2009, vol. 26, pp. 522–536. 7. Oremland, R.S., Stolz, J.F., and Hollibaugh, J.T., The Microbial Arsenic Cycle in Mono Lake, California, FEMS Microbiol. Ecol., 2004, vol. 48, pp. 15–27. 8. Oren, A., Enigma of Square and Triangular Halophilic Arhaea, in Enigmatic Microorganisms and Life in Extreme Environments, Dordrecht: Kluwer, 1999, pp. 339–355. 9. Oren, A., Microbial Life at High Salt Concentrations: Phylogenetic and Metabolic Diversity, Saline Systems, 2008, vol. 4, no. 2. doi:10.1186/1746144842/26 10. Takeuchia, M., Kawahatab, H., Guptab, L.P., Arsenic Resistance and Removal by Marine and NonMarine Bacteria, J. Biotechnol., 2007, vol. 127, no. 3, pp. 434– 442. 11. Winkler, D.W., An ecological study of Mono Lake, Cali fornia. Davis: Inst. Ecol. Publ., 1977, no. 12. 12. WolfeSimon, F., Blum, J.S., Kulp, T.R., A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus, Sciencexpress, www.sciencexpress.org/2December2010/ Page1/10.1126/science.1197258. 13. WolfeSimon, F., Davies, P.C.W., and Anbar, A.D., Did Nature Also Choose Arsenic?, Int. J. Astrobiol., 2009, vol. 8, no. 2, pp. 69–74.