The Great Oxidation Event expanded the genetic repertoire of arsenic metabolism and cycling Song-Can Chena,b,c,1, Guo-Xin Suna,1, Yu Yand, Konstantinos T. Konstantinidise,f, Si-Yu Zhange, Ye Denga, Xiao-Min Lia,c, Hui-Ling Cuia,c, Florin Musatb, Denny Poppg, Barry P. Rosenh,2, and Yong-Guan Zhua,i,2 aState Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 100085 Beijing, China; bDepartment of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany; cCollege of Resources and Environment, University of Chinese Academy of Sciences, 100049 Beijing, China; dDepartment of Environmental Science and Engineering, Huaqiao University, 361021 Xiamen, China; eSchool of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332; fSchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332; gDepartment of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, 04318 Leipzig, Germany; hDepartment of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199; and iKey Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 361021 Xiamen, China Edited by Donald E. Canfield, Institute of Biology and Nordic Center for Earth Evolution (NordCEE), University of Southern Denmark, Odense M., Denmark, and approved March 20, 2020 (received for review January 25, 2020) The rise of oxygen on the early Earth about 2.4 billion years ago under an atmosphere with very low oxygen levels (<<0.001% reorganized the redox cycle of harmful metal(loids), including that compared with present atmospheric level) (9). The rise of atmo- of arsenic, which doubtlessly imposed substantial barriers to the spheric oxygen (∼1% of present atmospheric levels) during the physiology and diversification of life. Evaluating the adaptive GOE between 2.4 and 2.3 Bya most likely led to intense oxidative biological responses to these environmental challenges is inher- weathering of arsenic-bearing minerals that liberated continental ently difficult because of the paucity of fossil records. Here we arsenic, predominantly as arsenate, for delivery to oceans from applied molecular clock analyses to 13 gene families participating rivers (3, 10). These processes would have resulted in the wide- in principal pathways of arsenic resistance and cycling, to explore spread appearance of oxidized arsenic species in the environment. the nature of early arsenic biogeocycles and decipher feedbacks We hypothesized that these dramatic shifts in the redox state of associated with planetary oxygenation. Our results reveal the arsenicals and their bioavailability imposed a strong selective advent of nascent arsenic resistance systems under the anoxic pressure on ancient microorganisms toward acquisition of novel environment predating the Great Oxidation Event (GOE), with the enzymatic systems conferring arsenic resistance. Current microbial primary function of detoxifying reduced arsenic compounds that fossil records lack the power to resolve the timing and causes of were abundant in Archean environments. To cope with the the origin of these tolerance and detoxification mechanisms. increased toxicity of oxidized arsenic species that occurred as Molecular and genetic studies have identified many arsenic oxygen built up in Earth’s atmosphere, we found that parts of resistance (ars) genes in extant organisms (SI Appendix, Table preexisting detoxification systems for trivalent arsenicals were S1). These include efflux permeases, redox enzymes, methyl- merged with newly emerged pathways that originated via conver- transferases, and transcriptional repressors. Arsenite efflux is gent evolution. Further expansion of arsenic resistance systems catalyzed by two evolutionarily unrelated groups of arsenite was made feasible by incorporation of oxygen-dependent enzy- matic pathways into the detoxification network. These genetic Significance innovations, together with adaptive responses to other redox- sensitive metals, provided organisms with novel mechanisms for The oxygenation of the atmosphere about 2.4 billion years ago adaption to changes in global biogeocycles that emerged as a remodeled global cycles of toxic, redox-sensitive metal(loids), in- consequence of the GOE. cluding that of arsenic, which must have represented a cataclysm in the history of life. Our understanding of biological adaptations arsenic | detoxification | evolution | oxygen | biogeochemistry surrounding this key transition remains unexplored. By estimating the timing of genetic systems for arsenic detoxification, we reveal ne of life’s earliest challenges was coping with the toxicity of an expansion of enzymes and pathways that accompanied ad- Oharmful metal(loids) (1). Understanding the nature and aptations to the biotoxicity of oxidized arsenic species produced timing of the onset of protective mechanisms is essential for the by Great Oxidation Event. These include enzymes originated via study of early evolution of Earth and life, yet limited information convergent evolution and pathways that use oxygen for enzy- is available. Arsenic is the most ubiquitous toxic metalloid in matic catalysis. Our results illustrate how life thrived under the nature, with two biologically relevant oxidation states: trivalent stress of metal(loid) toxicity and provide insights into environ- arsenite and pentavalent arsenate. Arsenite is generally more mental biogeochemical cycling and microbial evolution. toxic than arsenate, and perturbs the physiology of prokaryotes at micromolar levels (2, 3). Relatively high amounts (>20 μM) of Author contributions: B.P.R. and Y.-G.Z. designed research; S.-C.C., G.-X.S., X.-M.L., and H.-L.C. performed research; S.-C.C., Y.Y., K.T.K., S.-Y.Z., Y.D., and D.P. analyzed data; dissolved arsenic are nowadays frequently found in oceanic hy- and S.-C.C., G.-X.S., Y.Y., F.M., B.P.R., and Y.-G.Z. wrote the paper. drothermal vents or hot springs, environments that may have The authors declare no competing interest. conditions analogous to similar niches of primordial Earth. For This article is a PNAS Direct Submission. this reason, resistance pathways for transport and bio- This open access article is distributed under Creative Commons Attribution-NonCommercial- transformation of arsenic are believed to have emerged early in NoDerivatives License 4.0 (CC BY-NC-ND). the evolution of life on Earth (4–6). Environmentally, the rise of Data deposition: The sequence data used in this study were provided as supplementary atmospheric oxygen during the Great Oxidation Event (GOE) Datasets S1-S3. ∼ 2.4 billion years ago (Bya) is thought to have fundamentally 1 S.-C.C. and G.-X.S. contributed equally to this work. ’ changed arsenic chemistry in the Earth s surface and oceans (2, 2To whom correspondence may be addressed. Email: [email protected] or ygzhu@rcees. 7). Prior to the GOE, reduced arsenic species (i.e., arsenite) ac.cn. would have predominated over oxidized arsenics (i.e., arsenate) This article contains supporting information online at https://www.pnas.org/lookup/suppl/ because the atmosphere and oceans were anoxic and reducing (4, doi:10.1073/pnas.2001063117/-/DCSupplemental. 6, 8). Continental weathering of arsenic at this time is negligible First published April 29, 2020. 10414–10421 | PNAS | May 12, 2020 | vol. 117 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.2001063117 Downloaded by guest on September 27, 2021 efflux permeases: ArsB and Acr3 (11). Arsenate detoxification is set of six independent analyses were performed to evaluate the catalyzed by reductases (ArsC), with homology to the gluta- robustness of the results to prior assumptions of root age (analyses redoxin family (ArsC1), to low-molecular-weight phosphatases 1 and 7), subsampling of fossil calibrations (analyses 3, 4, 9, and (ArsC2), or by members of the CDC25 family of dual-specific 10), and alternative topologies (analyses 5, 6, 11, and 12). Median phosphatases (Acr2), respectively (12). These enzymes reduce gene ages under 12 analytical scenarios are shown in Tables 1 and intracellular arsenate to arsenite, the substrate of the two arse- 2, and the uncertainties associated with the results from all these nite efflux permeases. Additionally, arsenite can be methylated analyses were integrated over to provide composite credibility by ArsM, an arsenite S-adenosylmethionine (SAM) methyl- interval for each gene family (Fig. 2). Although the timing of transferase, to the more toxic species methylarsenite and dime- arsM and acr3 varied under different prior assumptions, all thylarsenite. In air, these are oxidized nonenzymatically to the analyses consistently recovered 95% credibility intervals en- largely nontoxic pentavalent species. However, methylarsenite tirely within the Archean eon, suggesting that they originated can be also detoxified by active extrusion from cells catalyzed by before the GOE. For arsC2, arsR1,andarsP,weestimatethat the methylarsenite-specific efflux permease ArsP (13), oxidation themediangeneagesarebeforeoratthebeginningofthe to methylarsenate by the methylarsenite-specific oxidase ArsH Paleoproterozoic period, with composite 95% confidence in- (14, 15), or demethylation to less toxic arsenite by the ArsI C-As tervals overlapping with the GOE. In contrast, arsB, arsI, arsH, lyase that cleaves the carbon–arsenic bond in methylarsenite arsR2, arsR3, arsC1,