Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation H. A. Meijer et al. Science 340, 82 (2013); DOI: 10.1126/science.1231197

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binds directly to the 40S ribosomal subunit and Translational Repression and only requires eIF3, eIF2, eIF5, eIF5B, and Met- tRNAi, whereas the encephalomyocarditis virus (EMCV) IRES requires all initiation factors apart eIF4A2 Activity Are Critical for from eIF4E (1).ACrPVIRESmRNAcontaining let-7 target sites is not repressed, unlike the con- MicroRNA-Mediated Gene Regulation trol mRNA (Fig. 1B), indicating that miRNA- mediated repression does not involve an elongation H. A. Meijer,* Y. W. Kong,* W. T. Lu,* A. Wilczynska,* R. V. Spriggs, S. W. Robinson, block, ribosome drop-off, or polypeptide degra- J. D. Godfrey, A. E. Willis, M. Bushell† dation. The HCV IRES reporter was also refrac- tory to miRNA-mediated repression, indicating MicroRNAs (miRNAs) control gene expression through both translational repression and that miRNA function is exerted before recruit- degradation of target messenger RNAs (mRNAs). However, the interplay between these processes ment of the 60S ribosomal subunit, the eIF2- and the precise molecular mechanisms involved remain unclear. Here, we show that translational tRNAiMet ternary complex, eIF3, eIF5, or eIF5B. inhibition is the primary event required for mRNA degradation. Translational inhibition depends on In contrast, translation of a let-7 reporter driven miRNAs impairing the function of the eIF4F initiation complex. We define the RNA helicase eIF4A2 by the EMCV IRES was repressed as efficient- as the key factor of eIF4F through which miRNAs function. We uncover a correlation between the ly as was cap-dependent translation, indicating presence of miRNA target sites in the 3′ untranslated region (3′UTR) of mRNAs and secondary that miRNA-mediated repression is exerted after structure in the 5′UTR and show that mRNAs with unstructured 5′UTRs are refractory to miRNA eIF4E and eIF4G recruitment. The observed ef- repression. These data support a linear model for miRNA-mediated gene regulation in which fects are not restricted to let-7 (fig. S3, A and B) translational repression via eIF4A2 is required first, followed by mRNA destabilization. and the IRESs are functional (fig. S3, C to F). Destabilization of the IRES-containing reporter icroRNAs (miRNAs) are noncoding pression is not dependent on deadenylation (fig. mRNAs only occurred on translationally repressed 21- to 25-nucleotide (nt) RNA mole- S2, F and G). Both of these mRNAs can bind one messages (Fig. 1C), even though nonrepressed Mcules that in metazoans base-pair im- poly(A)-binding protein (PABP) molecule (6); mRNAs remain associated with the targeting

perfectly with regions in target mRNAs [generally however, the former cannot be deadenylated (fig. miRNAs (fig. S4, A to C) and the RNA-induced on April 9, 2013 within the 3′ untranslated region (3′UTR)] and S2, H and I) (4, 7). Thus, the poly(A) tail, de- silencing complex (RISC) (fig. S4, D and E). repress the synthesis of the corresponding pro- adenylation, and mRNA degradation are not Additionally, we confirmed that the CrPV IRES teins (1). The mechanism for miRNA-mediated essential for miRNA-mediated translational re- reporter mRNA can undergo destabilization (fig. repression has remained elusive. Nevertheless, pression, which is in agreement with other ob- S4F). Thus, translational repression is the primary it is clear that miRNAs bind target mRNAs in servations (2–6, 8). and required event for miRNA-mediated control, complex with Argonaute proteins (Ago1 to Ago4 Evidence suggests that miRNAs exert their followed by mRNA destabilization. A number in humans). This complex then recruits one of the repressive activity at the translation initiation of factors may explain previous contradictory trinucleotide repeat–containing proteins (TNRC6A phase (9–12). To identify which translation ini- results involving miRNA-targeted IRES reporters,

to TNRC6C), and this in turn leads to both trans- tiation factors are required for repression, we ap- including the presence of a poly(A) tail and www.sciencemag.org lational repression and mRNA destabilization (1). pended the 5′UTRs of our reporter constructs method of mRNA delivery (1). To determine whether mRNA degradation with different internal ribosome entry site (IRES) Our analysis of IRES-dependent miRNA- is a cause or consequence of translational inhi- sequences (Fig. 1A). The cricket paralysis virus mediated repression excluded many of the trans- bition, we used transfection of in vitro transcribed (CrPV) IRES requires no initiation factors, binds lation initiation factors that might be implicated let-7 target reporter mRNAs. Translational repres- directly to the ribosomal subunits, and promotes in the repression mechanism. The initiation fac- sion of the target mRNA can clearly be observed elongation (1). The hepatitis C virus (HCV) IRES tors that are required for EMCV IRES– but not as early as 30 to 60 min after transfection in the absence of miRNA-dependent mRNA destabiliza- A Downloaded from tion (fig. S1), which is in agreement with (2–5). B As deadenylation occurs on miRNA-repressed mRNAs, we considered the requirement for the polyadenylate [poly(A)] tail in miRNA-mediated repression. Transfection of mRNAs with and with- out a poly(A) tail showed that unadenylated – mRNAs are efficiently repressedbymiRNAs,al- Fig. 1. Cap-dependent and EMCV IRES driven though the addition of a poly(A) tail increased translation is inhibited by let-7, whereas CrPV – the magnitude of repression (fig. S2, A and B). and HCV IRES driven translation is not. (A) Although increasing the length of the poly(A) Schematic representation of constructs used. Tran- scripts were either cap-dependent or driven by CrPV, tail results in more efficient translation rates (fig. HCV, EMCV IRESes T eight repeats of let-7 target sites S2, C and D), we observed maximal repression of ′ – in the 3 UTR. (B) HeLa cells were transfected with the C the let-7 targeted mRNA with a relatively short plasmids depicted in (A) together with a control firefly poly(A) tail (fig. S2E). Furthermore, we observed plasmid. Luciferase assays were performed equally efficient repression of an mRNA with a after 48 hours. Luciferase activity presented is Rluc/ poly(A) tail of exactly (A)25(C)10 as one with an Fluc, with the value for the constructs without miRNA (A)25 tail, confirming that miRNA-mediated re- target sites set at 100%. (C)RNAwasisolatedand analyzed by using quantitative reverse transcription Medical Research Council (MRC) Toxicology Unit, Hodgkin polymerase chain reaction (RT-PCR) in parallel to (B). Building, Lancaster Road, Leicester LE1 9HN, UK. Rluc mRNA levels were calculated relative to Fluc

*These authors contributed equally to this work. mRNA levels. levelsNormalized mRNA Normalized luciferase activity †Corresponding author. E-mail: [email protected]

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HCV IRES–driven translation are all components structs T let-7 target sites were used as readout. examine the early events in the miRNA repres- of the eIF4F complex. To confirm that eIF4F func- Only the knockdown of eIF4A2 affected miRNA- sion pathway, before mRNA destabilization, the tion (which includes helicase activity and tran- mediated repression (Fig. 2, C to E). This effect expression of either eIF4A2 or TNRC6A+B was script scanning activity) is essential for repression, was not specific to the particular eIF4A2 siRNA knocked down, followed by transfection of in we transfected mRNAs carrying either the b-globin used or restricted to one cell type (fig. S5, A to E). vitro transcribed mRNAs T let-7 target sites for 5′UTR or an unstructured (CAA)18 5′UTR, which Depletion of both eIF4As did not change the 1 hour. At this time point, siRNA depletion of does not require unwinding (13), T let-7 target extent of derepression (fig. S5, F and G). siRNA- eIF4A2 or TNRC6A+B had similar effects on sites (Fig. 2A). The mRNA bearing the unstruc- depletion of eIF4A2 also increased the expression the miRNA target (Fig. 2, G and H, and fig. S5I). tured 5′UTR was refractory to miRNA-mediated of a number of well-known endogenous miRNA Transfection of a dominant negative form of repression and even showed a slight activation targets (Fig. 2E and fig. S5H) (14). The knock- eIF4A1 inhibited translation but did not affect the (Fig. 2B), confirming a requirement for eIF4F, down of eIF4A2 or TNRC6A+B, but not eIF4A1, extent of miRNA-mediated regulation (fig. S6, A which is in agreement with (11, 12). resulted in stabilization of reporter mRNA with to C), whereas inhibition of both eIF4As with To identify which of the implicated eIF4F let-7 target sites (Fig. 2F), again showing the re- Pateamine A lead to a marked decrease in miRNA- components or associated factors are required for quirement for translational repression for mRNA mediated repression (fig. S6, D and E). Rescue miRNA-mediated translational repression, small destabilization. Together, these results identify the experiments showed that after depletion of interfering RNAs (siRNAs) were used to knock eIF4F component eIF4A2 as the primary and key eIF4A2, only eIF4A2 and not eIF4A1 can restore down their expression, and DNA reporter con- factor for miRNA-mediated gene silencing. To repression (Fig. 2, I and J, and fig. S6, F and G),

A E H on April 9, 2013

B Normalized translation rates

I www.sciencemag.org Normalized translation rates

C F

D Normalized luciferase activity Downloaded from

J Normalized mRNA levels Normalized mRNA

G Normalized luciferase activity

Fig. 2. eIF4A2 is the only component of eIF4F required for miRNA- of Western blot as indicated. IMP1 is a target of let-7. (F) HeLa cells were mediated repression. (A) Schematic representation of transcripts used for transfectedasin(D),RNAisolatedandanalyzedbymeansofquantitativeRT-PCR. transfection with either the b-globin 5′UTR or an unstructured (CAA)18 5′UTR T Rluc mRNA levels were calculated relative to Fluc mRNA. (G) Transcripts used for two let-7 target sites. (B) HeLa cells were transfected with the in vitro transcribed transfection, T eight let-7 target sites. (H) HeLa cells were transfected with the mRNAs depicted in (A). Protein and RNA were harvested after 90 min. Trans- indicated siRNAs. After 3 days, the cells were trypsinised, counted, and transfected lation rates were determined as a ratio of luciferase activity to mRNA levels (four with the in vitro transcribed mRNA depicted in (G). RNA and protein was collected biological repeats). (C) Constructs used for transfection in (D) to (F), T two let-7 after 60 min, and the translation rates were calculated (four biological repeats). (I) target sites. (D) HeLa cells were transfected with the indicated siRNAs or an LNA HeLa cells were transfected with the indicated siRNAs. Plasmids encoding the against let-7 for 48 hours. Next, cells were transfected with the plasmid indicated Flag-tagged proteins were transfected after 24 hours, and reporters as constructs depicted in (C) and the control. Luciferase activity depicted in (C) after another 24 hours. Luciferase assays were performed after a was assayed after 24 hours. Values for control reporters were set at 100%. (E) further 24 hours as in (D). (J) HeLa cells were transfected in parallel with (I). HeLa cells were transfected in parallel with (D). Lysates were analyzed by means Lysates were analyzed by means of Western blot as indicated.

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A CD

B Normalized mRNA levels Normalized mRNA

Fig. 3. eIF4A2 is preferentially recruited to miRNA-targeted mRNAs protein. After 48 hours, cells were lysed and subjected to Flag immu- and specifically associates with CNOT7, a component of the miRNA- noprecipitation followed by Western blot analysis. HC, heavy chain. (C) repression machinery.(A) Schematic representation of tethering constructs Experiment as in Fig. 3B, analyzed by means of quantitative RT-PCR. (D) with 12 repeats of the MS2 hairpin within the 3′UTR T eight let-7 target sites. Immunoprecipitation from untransfected HeLa cell lysates using specific (B) MS2 immunoprecipitation of let-7–repressed mRNAs shows greater asso- antibodies to eIF4A1 and eIF4A2 with ribonuclease (RNAse) A or with RNase ciation with eIF4A2. HeLa cells were transfected with the plasmid constructs inhibitor (SuperaseIn). Recovered proteins were analyzed by using Western depicted in (A) together with a plasmid-expressing Flag-tagged MS2 coat blot. Input, 10%.

confirming that eIF4A2 plays a critical role early in Fig. 4. mRNAs with miRNA the repression pathway independently of eIF4A1. target sites in their 3′UTR eIF4A1 and eIF4A2 are both adenosine 5´- have a greater degree of triphosphate (ATP)–dependent RNA helicases, secondary structure in the 5′UTR than do mRNAs believed to be involved in unwinding secondary on April 9, 2013 structure within the 5′UTR of mRNAs, thus al- without miRNA target ′ lowing the 40S ribosomal subunit to dock and sites.5UTR sequences were scan toward the start codon (1). These highly re- analyzed by using RNAfold lated factors share 90% similarity at the protein for potential secondary structure and grouped into level, with the major differences residing with- 10 kcal/mol-wide minimum in the N-terminal domain. Although eIF4A1 is free-energy (mfe) bins. mRNAs generally in excess of eIF4A2, they do not have within each bin were interro- identical functions (15). This prompted us to gatedforconservedmiRNA

explore the possibility that eIF4A2, but not target sites. mRNAs with www.sciencemag.org eIF4A1, was specifically interacting with micro- relatively short 5′UTRs (be- ribonucleoprotein complexes. To determine whether tween18and100nt)were endogenous eIF4A2 associated with miRNA- analyzed separately. Sta- repressed mRNAs, MS2 binding protein was used tistical significance for all to immunopurify mRNAs containing MS2 hair- mRNAs, P =1.84×10−197; pins T let-7 target sites within their 3′UTR. Western for short 5′UTR mRNAs, P = − blotting was used to identify associated proteins. 1.06 × 10 22.

This experiment clearly shows that eIF4A2 spe- Downloaded from cifically associates with let-7–repressed mRNAs pending on the presence or absence of conserved pression. The presence of eIF4A2 on a repressed (Fig. 3, A to C). Members of the TNRC6 pro- miRNA target sites in their 3′UTR as predicted mRNA via its interaction with the Ccr4-NOT com- tein family have been shown to interact with the with TargetScan (Fig. 4). mRNAs with miRNA plex would preclude the progression of initiation Ccr4-NOT complex to induce both translational target sites are more likely to have a greater de- directed by eIF4G and eIF4A1, both for EMCV repression and mRNA deadenylation (16–19). gree of secondary structure within their 5′UTR. IRES– and, more importantly, cap-dependent Because a recent structural study has shown that We reasoned that mRNAs with short 5′UTRs (less translation. cNOT1, a central component of this complex, con- than 100 nt) should be even more dependent on tains a MIF4G fold similar to the eIF4A binding secondary structure for miRNA-mediated activ- References and Notes site of eIF4G (20), we investigated whether eIF4A2 ity. Indeed, we observed a stronger relationship 1. R. J. Jackson, C. U. Hellen, T. V. Pestova, Nat. Rev. Mol. interacts with the Ccr4-NOT complex. Indeed, im- between structure and the presence of conserved Cell Biol. 11, 113 (2010). munoprecipitation of endogenous eIF4A2, but not miRNA target sites within short 5′UTRs (Fig. 4). 2. M. R. Fabian et al., Mol. Cell 35, 868 (2009). 3. S. Djuranovic, A. Nahvi, R. Green, Science 336, 237 (2012). of eIF4A1, reveals a specific RNA-independent as- Our data show that miRNAs induce gene 4. A. A. Bazzini, M. T. Lee, A. J. Giraldez, Science 336, sociationofeIF4A2withCNOT7(Fig.3D).We silencing with translational repression occurring 233 (2012). also observed that eIF4G is preferentially associ- first and being required for subsequent mRNA 5. J. Béthune, C. G. Artus-Revel, W. Filipowicz, EMBO Rep. ated with eIF4A1 (Fig. 3D), which was indepen- destabilization. Moreover, we demonstrate that 13, 716 (2012). ′ 6. F. Moretti, C. Kaiser, A. Zdanowicz-Specht, M. W. Hentze, dently confirmed (fig. S6H). eIF4A2 and structure within the 5 UTR of target Nat. Struct. Mol. Biol. 19, 603 (2012). Our results imply that mRNAs with functional mRNAs are critical for miRNA-mediated repres- 7. Y. Mishima et al., Proc. Natl. Acad. Sci. U.S.A. 109, 1104 miRNA-target sites in their 3′UTR would require sion. We show that eIF4A2 interacts with com- (2012). structured 5′UTRs to enable repression. To ex- ponents of the repression machinery, namely the 8. L. Wu, J. Fan, J. G. Belasco, Proc. Natl. Acad. Sci. U.S.A. ′ 103, 4034 (2006). amine this globally, 5 UTR structure prediction Ccr4-NOT complex. Interaction may be occurring 9. R. S. Pillai et al., Science 309, 1573 (2005). was performed by using RNAfold on the human via the MIF4G domain within cNOT1 (20)re- 10. R. Thermann, M. W. Hentze, Nature 447, 875 (2007). RefSeq database. The mRNAs were grouped de- cruiting eIF4A2 and imposing translational re- 11. G. Mathonnet et al., Science 317, 1764 (2007).

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12. A. Zdanowicz et al., Mol. Cell 35, 881 (2009). 19. A. Cooke, A. Prigge, M. Wickens, J. Biol. Chem. 285, Supplementary Materials 13. T. V. Pestova, V. G. Kolupaeva, Genes Dev. 16, 2906 (2002). 28506 (2010). www.sciencemag.org/cgi/content/full/340/6128/82/DC1 14. B. Boyerinas et al., Cancer Res. 68, 2587 (2008). 20. A. P. Petit et al., Nucleic Acids Res. 40, 11058 (2012). Materials and Methods 15. G. Galicia-Vázquez, R. Cencic, F. Robert, A. Q. Agenor, Supplementary Text J. Pelletier, RNA 18, 1373 (2012). Acknowledgments: We thank E. Smith, M. Stoneley, T. Pöyry, Figs. S1 to S6 16. M. Chekulaeva et al., Nat. Struct. Mol. Biol. 18, 1218 (2011). E. Jan, C. Jopling, T. Achsel, M. Hentze, and J. Pelletier for References (21–38) 17. J. E. Braun, E. Huntzinger, M. Fauser, E. Izaurralde, helpful comments and reagents. This work was funded by the MRC, Mol. Cell 44, 120 (2011). UK. M.B. is an MRC Senior Fellow, and A.E.W. is a Biotechnology 8 October 2012; accepted 31 January 2013 18. M. R. Fabian et al., Nat.Struct.Mol.Biol.18, 1211 (2011). and Biological Sciences Research Council Professorial Fellow. 10.1126/science.1231197

ceptors (Fig. 1 and table S1). Our finding extends Archaeal (Per)Chlorate Reduction at (per)chlorate reduction into the archaeal domain of life and high temperature environments. High Temperature: An Interplay of assays showed chlorate-reducing ac- tivity in cell-free extracts and suspensions of A. fulgidus VC-16 that were comparable with ac- Biotic and Abiotic Reactions tivities found in some mesophilic (per)chlorate- Martin G. Liebensteiner,1 Martijn W. H. Pinkse,2,3 Peter J. Schaap,4 reducing bacteria (table S2). An activity toward Alfons J. M. Stams,1,5* Bart P. Lomans6 perchlorate could not clearly be identified, which is similar to an earlier study on the (per)chlorate Perchlorate and chlorate anions [(per)chlorate] exist in the environment from natural and reductase of Azospira oryzae strain GR-1 (9). No anthropogenic sources, where they can serve as electron acceptors for bacteria. We performed chlorite activity could be detected, growth experiments combined with genomic and proteomic analyses of the hyperthermophile either. Consistent with this, no genes similar to Archaeoglobus fulgidus that show (per)chlorate reduction also extends into the archaeal domain of known chlorite dismutases were identified in life. The (per)chlorate reduction pathway in A. fulgidus relies on molybdo- that have thegenomeofA. fulgidus. On the other hand, no similarity with bacterial enzymes; however, chlorite is not enzymatically split into chloride and accumulation of chlorite was observed, suggesting

oxygen. Evidence suggests that it is eliminated by an interplay of abiotic and biotic redox reactions an alternative mechanism of chlorite conversion. on April 9, 2013 involving sulfur compounds. Biological (per)chlorate reduction by ancient archaea at high Perchlorate- and chlorate-grown cells of temperature may have prevented accumulation of perchlorate in early terrestrial environments and A. fulgidus are still able to use sulfate as elec- consequently given rise to oxidizing conditions on Earth before the rise of oxygenic photosynthesis. tron acceptor. When sulfate and perchlorate are present together in the cultures, both are used erchlorate and chlorate anions [together re- Proteobacteria (7). The widely accepted pathway simultaneously, whereas sulfate reduction is de- ferred to as (per)chlorate] in the environ- of biological (per)chlorate reduction consists of a layed if chlorate is present (Fig. 1, C and D). Pment have long been considered as arising two-step reduction from perchlorate, via chlorate Simultaneous perchlorate and sulfate reduction mainly from anthropogenic activities, namely the to chlorite, followed by a dismutation to molec- in a single culture is intriguing from an energetic

production of perchlorate-containing rocket pro- ular oxygen and chloride (8). This metabolism viewpoint. The midpoint potentials of redox www.sciencemag.org pellants and ammunitions followed by environ- relies on the action of a couples involved in (per)chlorate reduction are mental pollution (1). However, recent findings (Pcr) and a chlorate reductase (Clr), two functions high, whereas those involved in sulfate reduc- – – indicate that perchlorate is continuously formed that in perchlorate reducers are often performed by tion are low [for ClO4 /ClO3 ,redoxpotential 0 – – 0 naturally in the atmosphere, with proposed mech- a single enzyme (9); it further requires a chlorite (E ′)=+0.788V;ClO3 /ClO2 E ′ =+0.709V; – – 0 2– – 0 anisms ranging from photochemically triggered dismutase (Cld) to form molecular oxygen and ClO2 /Cl E ′ = +1.199 V versus SO4 /HSO3 E ′ = – – 0 processes (2) to electrical discharge–based reactions chloride. Microbial formation of molecular oxy- –0.516 V; HSO3 /HS E ′ = –0.110 V]. During and ozone oxidation of chlorides (3). Such natural gen under anaerobicity is a biochemical rarity. the reduction of (per)chlorate, the redox poten- sources make perchlorate an ubiquitous compound Because the reported mechanisms for natural tial is locally increasing, but the overall redox Downloaded from on Earth, although sizeable accumulations tend perchlorate generation on Earth seem to have state remains low in all the cultures throughout to be limited to certain arid environments, like the existed already during preanthropogenic times the entire experiment (≤ –200 mV), indicated by Atacama desert in Chile (4). Perchlorate deposits (1, 2), the appearance of biological reduction of resazurin in the medium and a redox electrode. also exist on Mars (5). It has been proposed that (per)chlorate may have been an important event Sulfide is normally omitted from media when the lack of perchlorate accumulation elsewhere in Earth’s history. One indication of an ancient growing (per)chlorate reducers but is used to es- on Earth might be due to microbial activity (4), origin for reduction of (per)chlorate would be its tablish the low redox potential required for growth which is supported by the widespread occurrence occurrence in microorganisms that thrive in envi- of strict anaerobes like A. fulgidus. Similar to of bacteria that can use perchlorate and chlorate as ronments resembling those of early Earth. Archaeo- sulfate reduction, (per)chlorate reduction in terminal electron acceptor for growth (6). globus fulgidus, a hyperthermophilic archaeon, A. fulgidus requires reduced conditions that are (Per)chlorate-reducing bacteria described fulfills this criterion. A. fulgidus was isolated established by the addition of sulfide to the me- so far belong mainly to the bacterial phylum of from marine hot vents close to Vulcano island in dium and even pronounced by sulfide formation Italy (10) but has since then been found in many if sulfate is present as well (Fig. 1, C and D). 1Laboratory of Microbiology, Wageningen University, Dreijenplein 2 extreme subsurface environments, such as hot oil Proteome analysis of cells grown with either 10, 6703 HB Wageningen, Netherlands. Analytical Biotech- reservoirs or geothermal formations (11, 12). It perchlorate or chlorate shows that, in comparison nology Section, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, Netherlands. is considered to be a major contributor to sulfate with sulfate-grown cells, there is an increased abun- 3Netherlands Proteomics Centre, Julianalaan 67, 2628 BC Delft, reduction and sulfide formation at high temperature. dance of a large number of proteins that are asso- Netherlands. 4Laboratory of Systems and Synthetic Biology, A. fulgidus strain VC-16 is the best-studied sulfate- ciated with redox and oxygen stress (table S3). We Wageningen University, Dreijenplein 10, 6703 HB Wageningen, reducing archaeon. Its genome contains many oxido- hypothesize that in the presence of (per)chlorate Netherlands. 5Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. 6Shell reductases genes with unknown function (13). these proteins play a crucial role in creating and Global Solutions International B.V., Kessler Park 1, 2288 GS We demonstrate that A. fulgidus strain VC-16 maintaining a low intracellular redox potential, Rijswijk, Netherlands. as well as A. fulgidus strain Z (14) (fig. S1) can which is a basic requirement for dissimilatory *Corresponding author. E-mail: [email protected] grow with perchlorate or chlorate as electron ac- sulfate reduction. The differential expression of

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