A C·As Lyase for Degradation of Environmental Organoarsenical

AC·As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

Masafumi Yoshinaga1 and Barry P. Rosen

Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199

Edited by Jerome Nriagu, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board April 16, 2014 (received for review February 18, 2014) Arsenic is the most widespread environmental toxin. Substantial More complex pentavalent aromatic arsenicals such as roxarsone amounts of pentavalent organoarsenicals have been used as herbi- [4-hydroxy-3-nitrophenylarsonic acid, Rox(V)] have been largely cides, such as monosodium methylarsonic acid (MSMA), and as used since the middle of the 1940s as antimicrobial growth pro- growth enhancers for animal husbandry, such as roxarsone moters for poultry and swine to control Coccidioides infections (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo envi- and improve weight gain, feed efficiency, and meat pigmentation ronmental degradation to more toxic inorganic arsenite [As(III)]. We (8, 9). These aromatic arsenicals are largely excreted unchanged previously demonstrated a two-step pathway of degradation of and introduced into the environment when chicken litter is ap- MSMA to As(III) by microbial communities involving sequential reduc- plied to farmland as fertilizer (8). Pentavalent organoarsenicals tion to methylarsonous acid [MAs(III)] by one bacterial species and are relatively benign and less toxic than inorganic arsenicals; demethylation from MAs(III) to As(III) by another. In this study, however, aromatic (8–10) and methyl (11, 12) arsenicals are de- the gene responsible for MAs(III) demethylation was identified from graded into more toxic inorganic forms in the environment, which Bacillus an environmental MAs(III)-demethylating isolate, sp. MD1. may contaminate the foods and water supplies. Although micro- arsI This gene, termed arsenic inducible gene ( ), is in an arsenic resis- bial degradation of environmental organoarsenicals has been ars tance ( ) operon and encodes a nonheme iron-dependent dioxyge- documented (8, 9, 11, 13), no molecular details of the reaction · nase with C As lyase activity. Heterologous expression of ArsI have been reported. We recently demonstrated that a microbial conferred MAs(III)-demethylating activity and MAs(III) resistance Escherichia coli community in Florida golf course soil carries out a two-step to an arsenic-hypersensitive strain of , demonstrat- pathway of MSMA reduction and demethylation (14). Here we ing that MAs(III) demethylation is a detoxification process. Purified + report the isolation of an environmental methylarsonous acid ArsI catalyzes Fe2 -dependent MAs(III) demethylation. In addition, [MAs(III)]-demethylating bacterium Bacillus sp. MD1 (for ArsI cleaves the C·As bond in trivalent roxarsone and other aromatic “MAs(III) demethylating”) from Florida golf course soil and the arsenicals. ArsI homologs are widely distributed in prokaryotes, and cloning of the gene, termed arsenic inducible gene (arsI), re- we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowl- sponsible for MAs(III) demethylation. The gene product, ArsI, is nonheme iron-dependent dioxygenase with C·As lyase activity. ArsI edge, this is the first report of a molecular mechanism for organo- · arsenic degradation by a C·As lyase. cleaves the C As bond in a wide range of trivalent organoarsenicals, including the trivalent roxarsone [Rox(III)], into As(III), which herbicide resistance | growth promoter degradation Significance he metalloid arsenic is the most common environmental toxic Tsubstance, entering the biosphere primarily from geochemical Organoarsenicals are used as herbicides, pesticides, antimicro- sources, but also through anthropogenic activities (1). Arsenic is bial growth promoters, and chemical warfare agents. Envi- a group 1 human carcinogen that ranks first on the Agency for ronmental organoarsenicals are microbially degraded, but the Toxic Substances and Disease Registry Priority List of Hazardous molecular mechanisms of breakdown are unknown. We pre- Substances (www.atsdr.cdc.gov/SPL/index.html). Microbial arsenic viously identified a two-step pathway of degradation involving sequential reduction and C·As bond cleavage. Here we transformations create a global arsenic biogeocycle (1). These bio- report cloning of the gene and characterization of the gene transformations include redox cycles between the relatively innoc- product for a C·As lyase, ArsI, a member of the family of type I

uous pentavalent arsenate and the considerably more toxic and SCIENCES extradiol dioxygenases. ArsI is the only enzyme shown to be

carcinogenic trivalent arsenite (2, 3). In addition, many microbes, ENVIRONMENTAL arsM involved in degradation of the reduced forms of the herbicide both prokaryotic and eukaryotic, have genes for inorganic monosodium methylarsonic acid and the antimicrobial growth arsenite [As(III)] S-adenosylmethionine methyltransferases that promoter roxarsone. As arsI genes are widely distributed in methylate inorganic As(III) to mono-, di-, and tri-methylated bacteria, ArsI-catalyzed organoarsenic degradation is proposed species (4, 5). The genes encoding arsenic transforming enzymes to have an impact on the arsenic biogeocycle. are widely distributed, and these arsenic biotransformations have been proposed to play significant roles in the arsenic biogeocycle Author contributions: M.Y. and B.P.R. designed research; M.Y. performed research; M.Y. and in remodeling the terrain in volcanic areas such as Yellowstone contributed new reagents/analytic tools; M.Y. and B.P.R. analyzed data; and M.Y. and B.P.R. National Park and regions of the world with high amounts of arsenic wrote the paper. in soil and water such as West Bengal and Bangladesh (3, 6). The authors declare no conflict of interest. Arsenicals, both inorganic and organic, have been used in This article is a PNAS Direct Submission. J.N. is a guest editor invited by the Editorial agriculture in the United States for more than a century (7). Board. Data deposition: The sequences reported in this paper have been deposited in the Gen- Historically, the use of inorganic arsenical pesticides/herbicides Bank database [accession nos. KF899846 (Bacillus sp. MD1 16S ribosomal RNA gene, par- has been largely replaced by methylated arsenicals such as tial sequence) and KF899847 (Bacillus sp. MD1, partial genome sequence)]. monosodium methylarsonic acid (MSMA), which is still in use as 1To whom correspondence should be addressed. E-mail: [email protected]. an herbicide for turf maintenance on golf courses, sod farms, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. highway rights of way, and for weed control on cotton fields (7). 1073/pnas.1403057111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1403057111 PNAS | May 27, 2014 | vol. 111 | no. 21 | 7701–7706 Downloaded by guest on September 27, 2021 strongly suggests that the environmental pentavalent phenyl- 2,3-dioxygenase encoded by akbC from Rhodococcus sp. strain arsenicals such as Rox(V) also undergo a two-step pathway of DK17 (20) and 2,3-dihydroxybiphenyl 1,2-dioxygenase encoded by sequential reduction and ArsI-catalyzed dearylation, in analogy bphC from Pseudomonas sp. strain KKS102 (21) (Fig. S5). Al- with the demethylation of MSMA by a microbial community. though the N- and C-terminal domains are structurally similar to Thus, ArsI-catalyzed C·As bond cleavage is a newly identified each other, only the C-terminal domain binds metal and functions mechanism for degradation of organoarsenical herbicides and in catalysis (19). The divalent metal binding site of these dioxy- antimicrobial growth promoters. genase contains a triad of three charged amino acid residues. A Results homology search using Protein Basic Local Alignment Search Bacillus Tool identified His5-His62-Glu115 as a putative metal binding site Isolation of a MAs(III)-Demethylating from Golf Course Soil. in the Bacillus ArsI, which corresponds to those of AkbC and We previously reported the isolation of methylarsonic acid Burkholderia BphC (Fig. S5). His5 is replaced by a glutamine residue in the [MAs(V)]-reducing bacterium sp. MR1 and the Thermomonospora curvata Streptomyces putative ArsI orthologs from DSM MAs(III)-demethylating bacterium sp. MD1 from Streptomyces coelicolor Florida golf course soil (14). Together, these two activities result in 43183 and A3 (2) (Fig. S5). degradation of the herbicide MSMA. In this study, a second A Basic Local Alignment Search Tool Link to Protein Align- bacterial strain capable of MAs(III) demethylation was isolated. ments and Structures search identified nearly 650 putative ArsI Like Streptomyces sp. MD1, this isolate demonstrated no MAs orthologs in 487 bacterial species, with no representatives in other Bacillus (V) transformation when cultured alone (Fig. S1A,curve2),but kingdoms. In the ArsI sequence there are four vicinal nearly completely transformed MAs(V) into As(III) when cocul- cysteine pairs. One of the four (Cys96-Cys97 in the Bacillus ArsI) tured with Burkholderia sp. MR1 (Fig. S1A, curve 1), suggesting that is conserved in all putative ArsI orthologs, and the other three the isolate possesses MAs(III) demethylating activity. In confirma- pairs in Bacillus ArsI, located near the C terminus, are not conserved tion, the isolate demethylated MAs(III) (Fig. S1B). The isolate was identified as a Gram-positive Bacillus by 16S ribosomal DNA sequence analysis (GenBank accession no. KF899846) (Fig. S2), and was designated Bacillus sp. MD1 [i.e., MAs(III) demethylating]. A 25000

Cloning the ArsI MAs(III) Demethylase. MAs(III) is more toxic than As(III) inorganic As(III) in Chang human hepatocytes (15) and hamsters 20000 (16). We showed that it is more toxic in Escherichia coli cells as well (Fig. S3) (17), suggesting that demethylation is a de- 15000 MAs(V) toxification process. By using the difference in toxicity between cps) MAs(III) and As(III), we cloned the gene responsible for MAs(III) MAs(III) demethylation. A genomic DNA library from Bacillus sp. MD1 1 was constructed and transformed into E. coli,andthetrans- 10000 formants were selected for MAs(III) resistance. Two MAs(III)- (c Arsenic 2 resistant clones were obtained, and their ability to demethylate 5000 MAs(III) confirmed. Because the pattern of HindIII-digested inserts of the two clones was identical, both probably have the same in- 3 sert, and one was chosen for further analysis. The restriction 0 fragment of ∼12 kbp was subcloned, and the region responsible 0 100 200 300 400 500 for MAs(III) demethylation was narrowed to approximately Time (sec) 4 kbp. This DNA was sequenced and found to contain five putative genes (Fig. S4). The first would encode a 99-residue ArsR As(III)- B 0.8 responsive transcriptional repressor except for an apparent frame shift that truncates the putative gene product after 72 0.7 residues. This is followed a putative gene similar to an ars gene of unknown function termed arsI (i.e., arsenic inducible gene) from 0.6 Cupriavidus metallidurans CH34 (18) (Fig. S5). The 161-residue ) 0.5 gene product has been annotated as a bleomycin resistance 600nm protein, glyoxalase or type I extradiol ring-cleaving dioxygenase. 0.4 Three other putative reading frames are encoded on the op- posite strand and annotated as encoding a FAD-dependent 0.3 monooxygenase and a GCN5-related N-acetyltransferase, more distantly, a partial sequence for a hemin ABC transporter (A Growth 0.2 ATP-binding subunit. Genes highly homologous to those three were rarely found in ars operons, indicating that these genes are 0.1 not involved in arsenic detoxification. In contrast, type I extradiol ring-cleaving dioxygenases catalyze cleavage of the C·C 0.0 bond of aromatic ring (19), and MAs(III) demethylation can be 0123456 predicted to involve cleavage of the analogous C·As bond, so it is MAs(III) conc. (μM) reasonable to propose that arsI is the responsible gene. Type I extradiol dioxygenases belong to the vicinal oxygen Fig. 1. ArsI confers MAs(III) demethylating activity and resistance in E. coli. Δ chelate superfamily and use a divalent metal ion to catalyze a (A) MAs(III) biotransformation by E. coli strain AW3110 ( ars) bearing plasmid pMAL-arsI (curve 1), vector plasmid pMAL-c2x (curve 2), or no cells reaction involving direct metal ion chelation by vicinal oxygens of 124 (curve 3) was analyzed by HPLC-ICP-MS (Materials and Methods). The x axis the substrate (19). Type I extradiol dioxygenases include two- represents column retention time and the y axis represents relative amounts domain and one-domain enzymes, and ArsI resembles a one- of arsenic expressed as counts per second (cps). (B) Growth of E. coli AW3110

domain enzyme. Most are two-domain proteins composed of N- bearing pMAL-arsI124 (●) or pMAL-c2x (▽) with the indicated concen- terminal and C-terminal domains exemplified by methylcatechol trations of MAs(III). Error bars represent the SD of three assays.

7702 | www.pnas.org/cgi/doi/10.1073/pnas.1403057111 Yoshinaga and Rosen Downloaded by guest on September 27, 2021 110 S6). Reductant is likely required to maintain the enzyme (cysteine 2+ 100 thiolates and Fe ) and the substrate MAs(III) in their reduced forms. Even in the presence of reductant, some MAs(III) oxidized 90

%) to MAs(V), probably nonenzymatically by air, but the remainder of 80 the MAs(III) was quantitatively transformed into As(III) over a period of 60 min (Fig. 3). 70 Members of the family of type I extradiol ring-cleaving dioxygenases 60 · acvity (% catalyze cleavage of C C bond of the aromatic ring by incorporating 50 an atom of molecular oxygen into each carbon of the target bond (19). O2 consumption by purified ArsI clearly increased when the 40 substrate MAs(III) was added (Fig. S7). The initial immediate 30 decrease in O2 was caused by the reducing reagent used to prepare -As lyase -As lyase

C MAs(III) and As(III). This supports the proposition that ArsI is 20 a dioxygenase that uses oxygen for cleavage of the C·As bond, in 10 which one oxygen atom from dioxygen is added to the arsenic and 0 the other is added to the carbon (Fig. S8). Although the organic No Fe2+ GSH Cysteine TCEP Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ product following MAs(III) cleavage has not been identified, it is addition GSH Cysteine TCEP GSH Cysteine Upper K TCEP TCEP predictedtobeformaldehyde(Fig. S8, ). The m for MAs(III) −1 was determined to be 2.9 ± 0.8 μM, with a kcat of 0.25 ± 0.02 min . + Fig. 2. Effect of Fe2 and reductants on the MAs(III)-demethylating activity The low turnover number for the enzymatic process of C·As bond of purified ArsI. The amount of As(III) produced upon demethylation of cleavage catalyzed by purified ArsI may reflect the fact that the μ μ 10 M MAs(III) by 1 M purified ArsI in each indicated condition was quan- enzyme, cofactor, and substrate are all oxygen-sensitive. In vivo, tified by HPLC-ICP-MS, as described in Materials and Methods. Activities are + expressed as the percentage of As(III) produced [4.0 μmol As(III)] with Fe2 , these may be protected by the low redox potential of the cytosol. cysteine, and TCEP. Error bars represent the SD of three assays. ArsI Cleaves the C·As Bond of Aromatic Organoarsenicals. The ability of ArsI to catalyze C·As bond breakage in more complex trivalent in all ArsI orthologs (Fig. S5). Conserved cysteine residues in arsenic aromatic organoarsenicals was examined. ArsI could degrade the resistance proteins often function as binding sites for trivalent Rox(III) to inorganic As(III) in vivo (Fig. 4A) and in vitro (Fig. arsenicals (22–24). The ArsI conserved Cys96-Cys97 cysteine 4B). With Rox(III) as substrate, assuming that ArsI is a ring- pair is located at the equivalent position as the aspartate residue cleavage dioxygenase, the organic product can be predicted to be used by AkbC and BphC to form their substrate binding sites 4-hydroxy-5-nitro-hexa-2,4-dienal (Fig. S8, Lower). Rox(III) is (Fig. S5). It is reasonable, therefore, to propose that Cys96 and more toxic than As(III) (Fig. S3) (17), and AW3110 cells bearing Cys97 form the MAs(III) binding site in ArsI. pMAL-arsI124 were resistant to Rox(III) (Fig. 4C), demonstrating Nonconserved cysteine pairs in other arsenic resistance proteins that cleavage of the C·As bond in Rox(III) is a detoxification such as ArsR, ArsD, and ArsM are not critical for the activity, and process. The values of Km and Kcat for Rox(III) degradation by − removal of these nonconserved cysteine residues improves protein ArsI were 6.4 ± 1.7 μMand0.22± 0.04 min 1, respectively, which production and crystallization (25–27). A truncated arsI was con- are compatible with the kinetic values for MAs(III) demethyla- structed lacking the sequence for residues from Glu125 to the C tion. ArsI-expressing AW3110 cells cleaved the C·As bond in the terminus, which included the three nonconserved cysteine pairs. reduced forms of other aromatic arsenical, including nitarsone A p This construct, designated ArsI124, was found to confer MAs(III) (4-nitrophenylarsonic acid; Fig. S9 )and -arsanilic acid resistance and demethylation (Fig. 1). E. coli strain AW3110, an (4-aminophenylarsonic acid; Fig. S9B). These results demon- arsenic-hypersensitive strain lacking the chromosomal arsRBC strate that ArsI is a generalized C·As lyase. operon (28), was transformed with plasmid pMAL-arsI124 expressing ArsI124 as a maltose-binding protein (MBP) fusion. This C- terminally truncated MBP fusion is henceforth termed ArsI for 6 simplicity. Cells expressing ArsI nearly completely transformed + ArsI MAs(III) into inorganic As(III) (Fig. 1A, curve 1), whereas no MAs(III) transformation occurred in control cells (Fig. 1A,curve2), 5 confirming that the single arsI gene encodes a C·As lyase that SCIENCES catalyzes demethylation of MAs(III). AW3110 cells bearing 4 plasmid pMAL-arsI124 were more resistant to MAs(III) than ENVIRONMENTAL cells bearing vector plasmid pMAL-c2x (Fig. 1B). These results support our hypothesis that transformation of MAs(III) to 3 As(III) is a detoxification process that confers herbicide resistance. It also demonstrates that the nonconserved C-terminal vicinal 2 cysteine pairs are not essential for enzymatic activity, and the As conc. μM truncated ArsI was used subsequently for this study. 1 Characterization of C·As Bond Cleavage. The requirements for demethylation activity of purified ArsI were examined (Fig. 2). Pu- 0 rified ArsI requires a ferrous ion and a reductant such as gluta- -10 0 10 20 30 40 50 60 2+ thione or cysteine for the C·As lyase activity. The activity with Fe Time (min) and cysteine was enhanced approximately eightfold by addition of tris the strong reducing reagent (2-carboxyethyl)phosphine (TCEP), Fig. 3. Time course of MAs(III) demethylation by purified ArsI. The time course and further assays were conducted in the presence of TCEP and of MAs(III) demethylation was conducted as described in Materials and Meth- 2+ cysteineinadditiontoFe . Some dioxygenases have enzymatic ods. The reaction was initiated by addition of ArsI (arrow). △, total arsenic; ○, 2+ activity with various metals (19), but ArsI uses Fe exclusively (Fig. MAs(III); ▼,MAs(V);and●, As(III). Error bars represent the SD of three assays.

Yoshinaga and Rosen PNAS | May 27, 2014 | vol. 111 | no. 21 | 7703 Downloaded by guest on September 27, 2021 majority of environmental arsenic not methylated? One reason is A 25000 that inorganic arsenic is renewed through geothermal sources. As(III) Another contributing factor is that organoarsenicals are contin- 20000 uously broken down by microbial transformations. In this report, we identify what is, to our knowledge, the first known enzyme, ArsI, for cleavage of the C·As bond. The gene for ArsI is found 15000 in many aerobic bacterial species that exist in communities in Rox(III) Rox(V) which methylating bacteria produce MAs(V) that reducing bac- c (cps) 1 teria transform into the more toxic MAs(III). ArsI-expressing 10000 bacteria detoxify the trivalent methylated species by trans-

Arsenic forming it back to inorganic arsenic. The continual interplay 2 creates a biogeocycle that maintains a balance between organic 5000 and inorganic species. During the past half century, mankind has upset that balance 3 by the introduction of massive amounts of organoarsenicals. 0 0 50 100 150 200 250 Methylated arsenicals such as MAs(V) and DMAs(V) (i.e., dime- Time (sec) thylarsinic acid) have long been used as herbicides and insect pesticides. Many of these are for weed control, especially for B 6 cotton, ornamental plants, lawns, and golf-course turf. Approx- + ArsI imately 3,000,000 lbs. of MAs(V), mainly as MSMA, and 100,000 5 lbs. of DMAs(V) have been applied annually in the United States (www.epa.gov/oppsrrd1/REDs/organic_arsenicals_red. 4 pdf). Aromatic compounds such as roxarsone, nitarsone, and

M) p-arsanilic acid have been used in animal husbandry for disease prevention, growth promotion, enhanced feed utilization, and 3 improved meat pigmentation. The majority of these compounds is not retained by the animals but excreted into the environment 2 unchanged. Based on broiler production and Rox(V) feed dosage, As conc. (μM it is estimated that ∼2,000,000 pounds of Rox(V) were released into environment in the United States annually by the poultry 1 industry alone (9). ArsI also catalyzes cleavage of the C·As bond in trivalent form 0 of these manmade aromatic arsenicals: roxarsone (Fig. 4), 0 10 20 30 40 50 60 nitarsone (Fig. S9A), and p-arsanilic acid (Fig. S9B). Degrada- Time (min) tion of such aromatic arsenicals has been demonstrated under C 0.8 aerobic (10) and anaerobic conditions (8–10). In analogy with the demethylation of MAs(V), we propose that these pentava- 0.7 lent aromatic arsenicals undergo a two-step pathway of sequential reduction and ArsI-catalyzed dearylation. We have not 0.6

) observed reduction of Rox(V) by the MAs(V)-reducing bacte-

0.5 rial isolates from Florida golf course soils, and so we propose

600nm that there are yet-unidentified bacteria capable of reducing Rox arsI 0.4 (V) to Rox(III). To date, all -carrying bacteria, including Bacillus sp. MD1, are aerobes. It is possible that alternate path- 0.3 ways exist in anaerobes. Although the use of MSMA in the United States is now re- Growth (A Growth 0.2 stricted to cotton fields, golf courses, sod farms, and highway rights of way, and roxarsone has been largely replaced by the 0.1 related nitarsone, these organoarsenicals are still produced and used in other countries such as India and China. Moreover, 0.0 0123456organoarsenicals have been used for chemical warfare for more Rox(III) conc.conc (μM) (μM) than a century. During the Vietnam War, the United States released more than 1.2 M gallons of DMAs(V), which was called “ ” Fig. 4. Degradation of and resistance to Rox(III). (A) Rox(III) biotransforma- Agent Blue, one of the rainbow herbicides to kill the rice, bam-

tion by E. coli AW3110 (Δars) bearing pMAL-arsI124 (curve 1), pMAL-c2x (curve 2), boo, and banana crops (29). Agent Blue was used until recently in or no cells (curve 3). (B) The time course of Rox(III) degradation by purified ArsI the United States for spraying of cotton fields and golf courses, was conducted as described in Materials and Methods. The reaction was ini- especially in Florida. Diphenylated arsenic compounds such as tiated by addition of ArsI (arrow). △, total arsenic; ○, Rox(III); ▼, Rox(V); and ●, Clark I (diphenylchloroarsine) and Clark II (diphenylcyanoarsine) As(III). (C) Resistance to Rox(III) was assayed by growth of E. coli AW3110 (Δars) were produced as chemical warfare agents during World Wars ● ▽ bearing pMAL-arsI124 ( ) or pMAL-c2x ( ). Error bars represent the SD of I and II. These were disposed of in land and seas after those wars. three assays. However, diphenylarsinic acid, which results from chemical transformation and accumulates in the environment, has been demonstrated to degrade into inorganic forms by bacterial isolates from Discussion contaminated sites (30). We speculate that bacteria with arsI genes The arsM gene for arsenic methyltransferases is widespread and are involved in the environmental degradation of these dimethyl and is found in members of every kingdom (1). This implies that diphenyl arsenicals. arsenic methylation is an ancient process occurring since the first The molecular mechanisms of MSMA reduction, the initial organisms arose nearly 3.5 billion years ago. Why, then, is the reaction of the two-step MAs(V) breakdown pathway, remains

7704 | www.pnas.org/cgi/doi/10.1073/pnas.1403057111 Yoshinaga and Rosen Downloaded by guest on September 27, 2021 unknown (14). Reduction of MSMA has been also observed in Plasmid DNA was extracted from two clones with MAs(III)-demethylating plants such as rice (31). As is the case with MSMA, the toxicity of activity, and the inserts were subcloned into vector plasmid pUC19 (New Rox(V) is also increased by reduction to Rox(III) (Fig. S3), and England BioLabs) using appropriate restriction enzymes (New England ArsI-catalyzed degradation of Rox(III) to As(III) confers arsenic BioLabs) and transformed into E. coli TOP10. The MAs(III)-demethylating C activity of the obtained subclones was analyzed by HPLC-ICP-MS as de- resistance (Fig. 4 ). We propose that the marked increase in scribed in SI Materials and Methods. By subcloning, the activity was nar- toxicity in the trivalent forms of these relatively nontoxic pen- rowed to a 4-kbp fragment, which was sequenced by primer-walking tavalent organoarsenicals is linked to bioactivation by reducing technique (GenBank accession no. KF899847). The arsI gene was cloned for organisms. To function as herbicides, pesticides, and antimicro- further investigation. CLUSTAL W (38) was used to build up multiple se- bial growth promoters, the pentavalent compounds must be re- quence alignment of ArsI homologs. duced to the active species (17). We analyzed the distribution of more than 100 arsI genes and ArsI Expression, Purification, and Characterization. The arsI gene product is found that all are in ars operons. There seems to be no specific predicted to have 161 aa residues, but, from multiple sequence alignment of ars gene associated with arsI,incontrasttoarsD and arsA, ArsI homologs, it appears that there is little sequence conservation in the which always go together because their functions are interrelated C-terminal 37 residues (Fig. S5). An arsI derivative encoding only the N-ter- (32). MerB is a C·Hg lyase that confers resistance to organo- minal 124 residues, designated arsI124, was amplified from Bacillus sp. MD1 genomic DNA by using forward primer GGAGGGAGAATTCATGAAATATG- mercurials (33). Although superficially similar to ArsI, MerB is an CGC (EcoRI site underlined) and reverse primer TGTGTAAGCTTTTATTCAAC- unrelated enzyme with a different reaction mechanism (34). Sim- AGTTGTC (HindIII site underlined). After digestion with EcoRI (New England ilarly, merB is usually associated with merA,andmer operons that BioLabs) and HindIII, the arsI gene was cloned into vector plasmid merB 124 include are mostly broad-spectrum resistances to inorganic pMAL-c2x (New England BioLabs), generating plasmid pMAL-arsI124 encoding and organomercurials (35, 36). In contrast, narrow-spectrum oper- a hybrid ArsI124 with an N-terminal MBP. The plasmids were transformed into ons that lack merB confer resistance to only inorganic mercury. the arsenic hypersensitive E. coli strain AW3110(DE3) Δars. The transformants In summary, to our knowledge, ArsI is the first and, at this point, were cultured in LB medium supplemented with 100 μg/mL ampicillin at only identified gene product involved in organoarsenic degradation. 37 °C to an absorbance at 600 nm of 0.6, at which point 0.1 mM isopropyl β-D-1-thiogalactopyranoside was added as inducer. After incubation at ArsI also expands the range of arsenic resistance from narrow −1 spectrum (inorganic) to broad spectrum (both inorganic and organ- 37 °C for an additional 4 h, the cells were transferred to ST 10 medium and incubated with 1 μM MAs(III), Rox(III), trivalent nitarsone, or trivalent oarsenicals). MerB-catalyzed organomercurial degradation pre- · arsI p-arsanilicacidat25°Cfor1h.CAs lyase activity was analyzed by HPLC-ICP-MS dominates in mercury-polluted sites (36). Because was cloned as described in SI Materials and Methods. To assay for resistance to arsenicals,

from a golf course treated with MSMA, we speculate that ArsI-cat- induced cells bearing vector pMAL-c2x or pMAL-arsI124 were washed once − alyzed organoarsenical degradation predominates in sites contami- with the same volume of ST medium (10-fold concentrated ST 10 1 me- nated with organoarsenicals, so ArsI degradation of organoarsenicals dium), diluted 50-fold into fresh ST medium supplemented with 50 μg/mL contributes to the arsenic biogeochemical cycle, completing the al- ampicillin and 0.2% D-glucose, and incubated with the indicated concen- ternation of arsenic methylation and demethylation. trations of indicated arsenicals at 30 °C for 4 h, following which growth was estimated from the absorbance at 600 nm.

Materials and Methods C-terminal truncated ArsI124 with an N-terminal MBP was purified as · Reagents. All chemicals were obtained from Sigma-Aldrich unless otherwise described in SI Materials and Methods.CAs lyase activity with purified ArsI mentioned. Rox(V) was purchased from Acros Organics. The trivalent forms of was assayed in a buffer consisting of 0.1 M morpholinopropane-1-sulfonic methylarsonic acid, roxarsone, nitarsone, and p-arsanilic acid were prepared acid (MOPS) and 0.15 M KCl, pH 7.0. To optimize the conditions, enzyme μ 2+ by a slight modification of the previous procedure (37). Briefly, 0.2 mM ar- (1 M) was incubated with or without 0.1 mM Fe , 1 mM reduced gluta- senical was mixed with 27 mM Na S O ,66mMNa S O , and 82 mM H SO , thione, 1 mM cysteine, and/or 3 mM TCEP, with the reaction initiated by 2 2 3 2 2 5 2 4 μ following which the pH was adjusted to 6 with NaOH. addition of 10 M MAs(III). After incubating at 37 °C with shaking at 200 rpm for 30 min, the reactions were terminated with EDTA, and the arsenic species Cloning the Gene for MAs(III) Resistance. A MAs(III)-demethylating bacterium were analyzed by HPLC-ICP-MS. The amount of As(III) in each sample was quantified from the corresponding peak area using Chromera software Bacillus sp. MD1 was isolated as described in SI Materials and Methods. The 2+ 2+ 2+ 2+ 2+ 2+ total DNA extracted from Bacillus sp. MD1 (14) was partially digested with (Perkin-Elmer). In other assays 0.1 mM Mg ,Ca ,Mn ,Co Ni ,Cu ,or Zn2+ were used in place of Fe2+. The best assay components were 1 mM HindIII (New England BioLabs) and ethanol-precipitated. The pelleted DNA 2+ was suspended in water and layered on a discontinuous sucrose gradient cysteine, 3 mM TCEP, and 0.1 mM Fe . For time-based assays, the reaction μ (10%, 20%, 30%, and 40%, wt/vol) containing 50 mM Tris, pH 8.0, 0.1 M was initiated by addition of 1 M ArsI to the reaction solution containing μ EDTA, and 0.1 M NaCl, and centrifuged at 210,000 × g for 5 h at 17 °C. After 5 M MAs(III) or Rox(III) and incubated at 37 °C with shaking at 200 rpm for centrifugation, the solution was carefully removed from the top of the layer 60 min. Reactions were terminated with EDTA at the indicated times and the and separated into fractions. Fractions containing DNA fragments of more arsenic species were analyzed by HPLC-ICP-MS as described in SI Materials than 2 kbp were combined and concentrated by ethanol precipitation. The and Methods. Each amount of the indicated arsenic species was quantified resulting DNA was ligated to vector plasmid pUC118 Hind III/BAP (Takara from the corresponding peak area. SCIENCES Bio), and the ligation mixture was transformed into E. coli strain TOP10 Kinetic analyses were performed with 1 μM ArsI and varying concen- (Invitrogen) by using a MicroPulser (Bio-Rad). trations of MAs(III) or Rox(III) up to 40 μM, and the amount of As(III) gen- ENVIRONMENTAL − The transformants were spread on three agar plates of ST 10 1 medium erated at 1 min was used to approximate initial rates. Kinetic constants were – (14) containing 50 μg/mL ampicillin, 0.2% D-glucose, and 0, 1, or 2 μM MAs(III). calculated by Hanes Woolf analysis (39). These plates were incubated at 30 °C until colonies formed. The plate with ForanalysisofO2 consumption, 7 μM ArsI was incubated in 0.1 M MOPS, pH 2+ the fewest number of colonies was replicated onto new plates containing 1, 7.0, with 0.15 M KCl, 10 μMFe , 0.5 mM cysteine until the base line level of O2 2, or 3 μM MAs(III) and incubated at 30 °C until colonies formed. Replicate consumption was constant. MAs(III) and As(III) were prepared by using reducing plating was repeated twice. The clones obtained from these selections were reagent as mentioned earlier. Then, 7 μM of the prepared MAs(III) or As(III), or

cultured in Luria–Bertani (LB) medium supplemented with 100 μg/mL am- reducing reagent only, was injected to initiate catalysis, and the change in O2 picillin at 37 °C overnight; then, the cultured cells were transferred to ST levels was monitored by using a 782 Oxygen Meter (Strathkelvin Instruments). − 10 1 medium supplied with 1 μM MAs(III) and incubated at 25 °C for 8 h. MAs(III) demethylation in the culture medium was analyzed by HPLC in- ACKNOWLEDGMENTS. This work was supported by National Institutes of ductively coupled plasma (ICP) MS as described in SI Materials and Methods. Health Grant R37 GM55425 (to B.P.R.).

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7706 | www.pnas.org/cgi/doi/10.1073/pnas.1403057111 Yoshinaga and Rosen Downloaded by guest on September 27, 2021