Biodegradation

Key Enzymes Enabling the Growth of Arthrobacter sp. Strain JBH1 with Nitroglycerin as the Sole Source of Carbon and Nitrogen

Johana Husserl, Joseph B. Hughes, Jim C. Spain
DOI: 10.1128/AEM.00006-12

ABSTRACT

Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG.

INTRODUCTION

Flavoproteins that catalyze the reductive elimination of nitrite from 1,2,3-trinitroglycerin (NG) (to form dinitroglycerol [DNG]) include pentaerythriol tetranitrate reductase (Onr) from Enterobacter clocae (16), glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter (23, 28), flavin oxidoreductase (YqjM) from Bacillus subtilis (14), and xenobiotic reductases (XenA and XenB) from Pseudomonas putida and Pseudomonas fluorescens (6). All of the enzymes are members of the old yellow enzyme (OYE) family (23, 33), require flavin mononucleotide (FMN), use NADPH as an electron donor, and catalyze the reductive elimination of nitrate ester groups from NG with concomitant release of nitrite (7, 15, 23, 33). In all cases, both 1,2- and 1,3-DNG are produced, although selectivity for attack at either C1 or C2 has been observed in some cases (7, 28). There is less information about the enzymatic removal of the second nitrate ester group, although there is evidence that a single enzyme can sequentially catalyze the elimination of both the first and second nitrate ester groups (7). Both 1-mononitroglycerol (1-MNG) and 2-MNG are produced as a result of the reduction process. In the case of XenA, enzymatic regioselectivity results in the preferential production of 1-MNG (7). Although there is evidence of the complete conversion of NG to glycerol by bacterial cell extracts (24), the reduction rate of MNG by OYEs is 4 orders of magnitude slower than that of NG, and assays conducted with purified enzymes suggest that a different enzyme may be required to convert MNG to glycerol in vivo (7).

Arthrobacter sp. strain JBH1 was isolated based on its ability to grow on NG as the sole source of carbon and nitrogen (19). The discovery of bacteria that mineralize NG provides a foundation for potential remediation strategies that involve biodegradation and natural attenuation at NG-contaminated sites. However, the mechanism used by JBH1 to grow on NG is still unknown. Here we describe the NG degradation mechanism in JBH1 and the genes encoding the enzymes involved in the pathway. The key enzyme that allows productive metabolism of NG appears to be a kinase that catalyzes the ATP-dependent phosphorylation of the 1-MNG that is produced by a flavoprotein reductase.

MATERIALS AND METHODS

Chemicals.NG, 1,2-DNG, 1,3-DNG, and 1-MNG standard solutions in acetonitrile were purchased from Cerilliant (Round Rock, TX). NG was synthesized as previously described (31).

Analytical.NG, 1,2-DNG, 1,3-DNG, 1-MNG, and 2-MNG were quantified using an Agilent 1100 high-performance liquid chromatograph (HPLC) equipped with a Supelco LC-18 column (250 by 4.6 mm, 5 μm) and a UV detector. Methanol-water (50% [vol/vol] or 7% [vol/vol]) was used as the mobile phase at a flow rate of 1 ml/min, and absorbance was monitored at 214 nm. Nitrite was qualitatively analyzed using a colorimetric method, 4500-NO2− B (11), and quantitative measurements of nitrite and nitrate were conducted with a Dionex ion chromatograph equipped with an IonPac AS14A anion-exchange column (Dionex, CA). The eluents were sodium carbonate (8 mM) and sodium bicarbonate (1 mM) at a flow rate of 1 ml/min. Total protein was quantified using a Micro BCA (bicinchoninic acid) protein assay kit (Pierce Biotechnology, IL). HPLC-mass spectrometry (MS) analysis was conducted by the Georgia Institute of Technology Bioanalytical Mass Spectrometry Facility (Atlanta, GA).

Growth of JBH1.Arthrobacter JBH1 was grown on Luria-Bertani (LB) broth at room temperature or on minimal medium (30) supplemented with NG (0.26 mM).

Genome sequencing and annotation.Total DNA from JBH1 was isolated using an UltraClean microbial DNA isolation kit (Mobio Laboratories, Inc., CA) following the manufacturer's directions. 454 pyrosequencing was done by the Emory GRA Genomic Center (Atlanta, GA), and the results were analyzed using the CLC Genomics Workbench (5). The RAST (Rapid Annotation using Subsystem Technology) Prokaryotic Annotation Service (3) was used to annotate the draft genome and to identify putative members of the OYE family.

Fosmid DNA sequencing and in silico analysis.A CopyControl fosmid library production kit (Epicentre Biotechnologies, Madison, WI) was used to construct a fosmid library with DNA from JBH1, following the manufacturer's instructions. Fosmids were screened for the ability to release nitrite when 1-MNG was added to the culture. Fosmid pJBH1A3 DNA (Table 1) was purified with a FosmidMAX DNA purification kit (Epicentre Biotechnologies,WI), and 454 pyrosequencing was done by the Georgia Genomics Facility (Athens, GA). The reads were assembled with CLC Genomics Workbench (5).

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Table 1

Bacterial strains, plasmids, and primers used in this study

Transposon mutagenesis and screening.An EZ-Tn5 <oriV/KAN-2> insertion kit (Epicentre Biotechnologies, Madison, Wisconsin) was used for transposon mutagenesis of the fosmid clone pJBH1A3. Colonies grown on LB agar with chloramphenicol (34 mg/liter) and kanamycin (50 mg/liter) were transferred to 96-well plates containing LB medium with chloramphenicol (34 mg/liter), kanamycin (50 mg/liter), and CopyControl fosmid induction solution (Epicentre Biotechnologies, Madison, WI). Cultures were incubated at 37°C with shaking (250 rpm) for 16 h. Samples (50 μl) from each well were transferred to a second 96-well plate containing 1-MNG (73 μM) in phosphate buffer (26 mM [pH 7.2]) and incubated for 16 h at room temperature. Suspensions containing fosmid clones were screened colorimetrically for nitrite release from 1-MNG. Mutants that did not release nitrite from 1-MNG were sequenced using Ez-Tn5-specific outward-reading primers (Ez-Tn5 KAN-2 FP-1 and Ez-Tn5 KAN-2 RP-1; Epicentre Biotechnologies, Madison, WI) by Genewiz, NJ.

Cloning and overexpression of genes (pfvA, pfvB, pfvC, pfvD, and mngP).DNA was amplified by PCR using the appropriate primers (Table 1) (Integrated DNA Technologies, Coralville, IA) and TaKaRa Ex Taq polymerase (TaKaRa Bio USA, Madison, WI). The purified PCR products were ligated into NdeI and HindIII (pfvA) or EcoRI and HindIII (pfvB, pfvC, pfvD, and mngP) sites of the pET-24a vector (Invitrogen Corp., Carlsbad, CA). The resulting recombinant plasmids (Table 1) were transformed into Escherichia coli DH5α (New England BioLabs, Ipswich, MA) to maintain the plasmid or into E. coli Rosetta 2(DE3) competent cells (Novagen) (Table 1) for overexpression according to the manufacturers' instructions.

For overexpression, single colonies of E. coli Rosetta 2(pJBH1-1), Rosetta 2(pJBH1-2), Rosetta 2(pJBH1-3), Rosetta 2(pJBH1-4), or Rosetta 2(pJBH1-MNGK) were transferred into LB medium supplemented with kanamycin (50 mg/liter) and chloramphenicol (34 mg/liter) and incubated for 3 h at 37°C with shaking. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (200 mg/liter) was added, and cultures were incubated at 37°C with shaking for 12 h. Cultures containing plasmids pJBH1-1 to pJBH1-4 were centrifuged at 4°C and washed (three times) with phosphate buffer (26 mM [pH 7.2]). Rosetta 2(pJBH1-MNGK) was washed with glycine buffer (100 mM [pH 8.3]) after centrifugation. Overexpression of the enzymes was verified by polyacrylamide gel electrophoresis.

Enzyme assays.Cells were lysed using a French pressure cell at 20,000 lb/in2, and cell debris was removed by centrifugation (20,000 × g, 4°C, 60 min). Enzyme assays were conducted at room temperature. NG oxidoreductase was assayed in phosphate buffer (26 mM) containing cell extract (100 μg protein/ml) and NG (70 μM). NADPH (140 μM) was added to the mixture 10 min after the start of incubation. Samples were analyzed at appropriate intervals for NG, DNG, and MNG by HPLC. MNG kinase was assayed in glycine buffer (100 mM [pH 8.3]) containing cell extract (100 mg/ml) and MNG (∼70 μM). ATP (100 μM) and MgCl2 (250 μM) were added 10 min after the start of incubation, and the intermediates were analyzed at appropriate intervals by HPLC and ion chromatography. Changes in the 1-MNG concentration were used to determine transformation rates.

RESULTS

Transformation by JBH1 cell extracts.Comparison of NG degradation activity in cells grown with and without NG indicated that the NG degradation pathway in JBH1 is constitutive (data not shown). Crude extracts from JBH1 catalyzed the NADPH-dependent conversion of NG to 1,2-DNG and 1-MNG (Fig. 1A) (0.46 μmol NG/h/mg protein) with concomitant release of nitrite, which is consistent with previous reports of NG transformation catalyzed by OYEs (6, 7, 23, 33). The absence of 1,3-DNG and 2-MNG was consistent with our previous observation (19) that the initial steps in the pathway leading to the formation of 1-MNG are highly specific. Reaction mixtures without added cofactors or with NADPH did not transform 1-MNG.

Fig 1
Fig 1

Transformation of NG and MNG by extracts of JBH1. (A) Transformation of NG after addition of NADPH (200 μM); (B) 1-MNG transformation after addition of MgCl2 (250 μM) and ATP (100 μM) (protein concentration, 100 μg/ml).

The accumulation of 1-MNG indicated that the activity of a second enzyme system was required for subsequent metabolism of 1-MNG. When ATP was added to cell extracts from JBH1, 1-MNG was transformed without the release of nitrite or nitrate (Fig. 1B).

Four genes (pfvA, pfvB, pfvC, and pfvD) related to OYEs known to transform NG were found in the genome of JBH1. The relatedness to OYEs known to denitrate NG (Table 2) is within the range of identities (29 to 48%) among OYEs known to transform NG (XenA, XenB, Onr, NerA, and YqjM). Both the substrate binding sites and FMN binding sites are conserved.

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Table 2

Comparison of amino acid sequences of putative OYEs from JBH1 with those of OYEs known to denitrate NGa

To investigate whether any of the putative OYEs identified in the JBH1 genome are responsible for the initial transformation of NG to 1-MNG, pfvA, pfvB, pfvC, and pfvD, were cloned and overexpressed. Controls carrying the empty pET24a vector catalyzed the rapid denitration of NG to 1,2- and 1,3-DNG but did not produce substantial amounts of 1-MNG, the major denitration intermediate observed in JBH1 cell extracts (above). Clones containing pfvA, pfvB, pfvC, and pfvD converted NG to dinitroglycerin slightly faster than the control (data not shown). Only the clone expressing pfvC produced substantial amounts of 1-MNG (Fig. 2). The results indicate that NG oxidoreductase, PfvC, catalyzes the formation of 1-MNG in JBH1. The closest relative (85% amino acid identity to PfvC) is NADH:flavin oxidoreductase from Arthrobacter phenanthrenivorans Sphe3 (accession no. NC-015145.1). The context of pfvC in JBH1 (Fig. 3) is identical to that observed in two other Arthrobacter strains (Arthrobacter aurescens TC1 and Arthrobacter sp. strain FB24) and very similar to that observed in Renibacterium salmoninarum (4). None of the above homologs have been functionally annotated.

Fig 2
Fig 2

1-MNG produced by E. coli cells (optical density at 600 nm [OD600] = 0.8) expressing putative NG oxidoreductases after transformation of NG (290 μM) in 10 h. Bars indicate average ± range of duplicates.

Fig 3
Fig 3

Context of pfvC compared to similar arrangements in A. JBH1, A. aurescens TC1, Arthrobacter sp. strain FB24, and R. salmoninarum ATCC 33209. Arrows: 1, NADH flavin oxidoreductase; 2, thioredoxin domain-containing protein EC-YbbN; 3, putative ABC transporter/ATP-binding protein; 4, putative integral membrane transport protein; 5, hypothetical protein; 6, hypothetical protein, nicotinamidase.

Transformation of MNG.Because 1-MNG transformation was stimulated by ATP and not NADPH (Fig. 1B), we tested the hypothesis that a kinase similar to glycerol kinase might catalyze the next step in the catabolic pathway (Table 3). Enzyme assays with extracts from JBH1 showed a 3-fold increase in 1-MNG transformation rate (compared to Fig. 1B) when the initial ATP concentration was increased from 100 to 200 μM and a 5-fold increase when the initial 1-MNG concentration was increased to 150 μM. The maximum initial rate (45 nmol/min/mg protein) was sufficient to account for the rate of 1-MNG transformation by intact cells (30 nmol/min/mg protein). When magnesium was omitted from the assay mixture, the transformation rate was reduced by over 40%, which would be similar to the properties of glycerol kinase (17). Also consistent with glycerol kinase, ADP is highly inhibitory to the reaction; in the presence of ATP (100 μM), the addition of ADP (100 μM) completely inhibited the MNG transformation. The observation is consistent with the rapid decline in the transformation rate after the first few minutes (Fig. 1). No nitrite or nitrate was detected in any of the reaction mixtures. One unknown metabolite detected by HPLC increased in concentration as 1-MNG disappeared. LC-MS analysis revealed a compound with a molecular ion at m/z 215.7, (M−-H) and a major fragment at m/z 153 (M−-ONO2). The mass spectrum is consistent with the structure of 1-nitro-3-phosphoglycerol. The results indicate that transformation of 1-MNG is not catalyzed by an old yellow enzyme but by a kinase.

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Table 3

Transformation of 1-MNG in JBH1 cell extracts under different experimental conditions

A fosmid library was screened for clones able to transform MNG. One clone containing the plasmid pJBH1A3 transformed MNG (up to 0.15 μmol/h mg protein) with concomitant release of nitrite. The 34-kb fosmid insert, pJBH1A3 (GenBank accession no. JQ671543), was purified and sequenced.

Transposon mutagenesis of pJBH1A3 produced several clones without the ability to transform 1-MNG. Sequencing of the flanking regions indicated that interrupting either of two genes eliminated the transformation of MNG (see Fig. S1 in the supplemental material). orf11 encodes an FIC (filamentation induced by cyclic AMP [cAMP]) motif (22), and orf14 encodes a glycerol kinase homolog. The two open reading frames (ORFs) are about 3.3 kb apart. The region following orf11 contains a potential hairpin structure which could act as an intrinsic translation terminator (34). The distance between orf11 and orf14 (3.3 kb) and the presence of the intervening translation terminator suggest that the genes are in different operons.

The protein encoded by orf14 was given the designation mononitroglycerol kinase (MngP). The closest biochemically characterized enzyme to MngP is a glycerol kinase (GlpK) from Cellulomonas sp. (1) (67% identity based on amino acids). E. coli clones containing mngP transformed 1-MNG with release of nitrite, whereas controls containing the empty pET24a vector did not (data not shown).

The maximum activity of MngP was in the alkaline range, although spontaneous hydrolysis of 1-MNG at high pH prevented the determination of the pH optimum (Fig. 4). Similar kinases, including those from E. coli and Cellulomonas sp., have pH optima higher than 9.3 (17, 29). Glycerol kinase from Cellulomonas sp. is commercially available and was tested for its ability to transform 1-MNG. No transformation was observed under the experimental conditions used.

Fig 4
Fig 4

1 MNG transformation rate in cell extracts from Arthrobacter sp. strain JBH1, E. coli cells with expressed MngP, and the E. coli control.

orf11 encodes a 252-amino-acid protein and contains an FIC domain (1). The closest characterized enzyme is BepA from Bartonella henselae (25), a type IV secretion system effector (30% identity). The FIC motif [HPFx(D/E)GN(G/K)R] is widely distributed (35), and it was recently reported that some FIC domain proteins use ATP to catalyze the addition of AMP to other proteins in a process known as “AMPylation” (35). AMPylation is a posttranslational modification mechanism similar to phosphorylation, in which the added AMP moiety changes the activity of the protein (20, 22). Efforts to overexpress orf11 in E. coli and to determine its role in NG degradation were unsuccessful.

The understanding of the physiological role of FIC homologs is in the early stages (20, 22, 35). The role of the orf11-encoded protein in 1-MNG catabolism is unknown, although it might be involved in the regulation of mngP, which would be consistent with the proposed role in other systems (20). Because the FIC homolog is in a separate operon from mngP, it is unlikely that inactivation of orf11 could affect the translation of mngP in vitro. The fact that MngP catalyzed the transformation of 1-MNG suggests that the FIC homolog expressed in JBH1 does not play a direct catalytic role in the transformation of MNG.

DISCUSSION

It is clear that an NADPH-dependent enzyme in JBH1 can catalyze the reduction of the first and second nitrate ester groups from NG but that the nitrate ester of 1-MNG cannot be removed by the same reductive mechanism. The observations are consistent with previous results (7) indicating that transformation of MNG by OYEs is several orders of magnitude slower than transformation of NG, thus making it the bottleneck for growth on NG as the sole source of carbon. It is likely that the slower rate of MNG transformation catalyzed by flavoproteins is associated with differences in polarity or interaction with the active site of the oxidoreductase.

Although the results indicate that PfvC is responsible for the production of 1-MNG, they do not rule out the possibility that other enzymes might be involved in the initial transformation. Background reductase activity in E. coli (7) precluded obtaining a rigorous mass balance and distinguishing the contribution of the enzymes in E. coli from that of the overexpressed enzyme. Structural and mechanistic studies will be required to explain how the enzyme produces 1-MNG via 1,2-NG and avoids accumulation of the unproductive 2-MNG. They should also reveal the reason for the lack of activity with 1MNG.

A key finding of this study is the ATP-dependent transformation of 1-MNG by a glycerol kinase homolog in JBH1 cell extracts. The widely studied glycerol kinases (GKs) (2, 9, 18, 26, 27, 36, 37) transfer a phosphate group from ATP to glycerol and produce glycerol-3-phosphate. The glycerol-3-phosphate is then oxidized to dihydroxyacetone phosphate, which enters glycolysis/gluconeogenesis to be used as a carbon source (26, 27). The C-3-substituted glycerol analogs 3-chloro-1,2 propanediol, 3-fluoro-1,2 propanediol, 3-butene-1,2-diol, 1,2,4-butanetriol, and 3-mercaptopropane-1,2-diol can also be transformed by glycerol kinases (10, 12). To our knowledge, there are no reports of nitroester transformation by similar enzymes. Glycerol kinases are less tolerant toward C-2-substituted propanediols (10, 12), which could explain our previous observation that JBH1 cannot grow on 2-MNG (19). The above observation emphasizes the importance of the apparent specialization of PfvC to selectively produce 1-MNG; if 2-MNG was produced, the pathway involving a glycerol kinase would not be possible.

Based on the known mechanisms used by glycerol kinases to transform substituted glycerols, two transformation pathways can be envisioned for conversion of 1-MNG. In the first potential pathway, the carbon in the 3 position is phosphorylated and the nitrite is released by subsequent unknown reactions (Fig. 5). In the second pathway, the carbon that contains the nitroester bond is phosphorylated, directly displacing the nitrate ester and releasing nitrate. The phosphoglycerol would readily enter the central metabolism. In either scenario, glycerol could not be an intermediate.

Fig 5
Fig 5

Proposed transformation of NG to 1-nitro-3-phosphoglycerol.

The lack of nitrite or nitrate release during the transformation of 1-MNG by cell extracts and the detection of a product with mass spectral properties consistent with 1-nitro-3-phosphoglycerol strongly support the first hypothesis. It thus seems likely that enzymes of central metabolism can catalyze the subsequent assimilation of 1-nitro-3-phosphoglycerol in JBH1. The fact that nitrite was released from 1-MNG by resting cells of E. coli overexpressing mngP indicates that the E. coli host also contains an enzyme or enzymes that catalyze release of nitrite from 1-nitro-3-phosphoglycerol. The identity of the enzyme(s) is the subject of current research.

Despite the high identity between MngP from JBH1 and glycerol kinases (Cellulomonas sp., 67%; E. coli, 52%), a sequence alignment indicated significant differences at the glycerol binding site. A conserved glutamine (E84 in E. coli and E82 in Cellulomonas sp.) and tyrosine (Y135 in E. coli and Y133 in Cellulomonas sp.) are replaced by alanine and valine in MngP. The smaller side chains of alanine and valine would allow more room for the binding of the larger 1-MNG molecule in the active site. The details of the reaction mechanism remain to be established after purification of the enzyme.

Differences at the posttranslational modification sites involved in regulation are also evident. For instance, MngP lacks the histidyl residue that is commonly phosphorylated to activate glycerol kinases from Gram-positive bacteria (36). Likewise, the region that binds to IIIGlc in E. coli is not present in MngP. The observations suggest that a mechanism distinct from those of known glycerol kinases is involved in the regulation of MngP. It should be noted that MngP appears to be constitutive in JBH1, which could enable it to function in a new pathway without the regulatory constraints of the system from which it was recruited (13).

The region in the genome containing mngP (see Fig. S1 in the supplemental material) (12,000 to 18,000 bp) has a lower G+C content than the surrounding DNA. Additionally, several genes that encode resolvases and transposases are located within a few kilobases of mngP (data not shown), which suggests that mngP was recently acquired by horizontal gene transfer. None of the MngP homologs have been reported to transform nitroesters.

During evolution of catabolic pathways for xenobiotic chemicals, recruitment of genes and evolution of new substrate specificities often precede optimization of regulation (8, 21). The constitutive expression of pfvC and mngP along with the slow growth of the Arthrobacter on NG suggest that the catabolic pathway is in the early stages of evolution. A plausible scenario for the sequence of events would be the initial involvement of the OYE, which would allow NG to serve as a nitrogen source for Arthrobacter as it does in several other strains (7, 16, 32). The existence of pfvC homologs in a very similar context in other Arthrobacter strains suggests that pfvC was already present in the progenitor of strain JBH1. Recruitment of mngP, either from the genome of JBH1 or by horizontal gene transfer, would extend the pathway to produce an intermediate, 1-nitro-3-phosphoglycerol, from which the final nitrate ester could be eliminated. JBH1 relies on enzymes of central metabolism to assimilate the carbon from 1-nitro-3-phosphoglycerol, as indicated by its disappearance and concomitant release of nitrite in E. coli clones containing mngP. Constitutive expression of the initial enzymes would provide the basis for selection of mutant strains with improved substrate specificities that would support growth on NG.

The above scenario describing recent and ongoing evolution of the pathway is supported by the fact that bacteria able to grow on NG as the sole source of carbon, nitrogen, and energy have been sought for many years without success. Previous isolation of bacteria able to use NG as a nitrogen source suggests that the oxidoreductase homologs are widely distributed. The long history of NG contamination in the soil from which the inoculum was obtained along with the extended selective enrichment in the laboratory (19) provided ample opportunity for recruitment of mngP, enhancement of the catalytic properties of the two key enzymes, and finally the emergence of a strain able to grow on NG. The growth rates are still slow, which suggests considerable opportunity for continued evolution of the catabolic pathway and for advances in understanding as the process unfolds in the laboratory.

An OYE that selectively produces 1-MNG and a glycerol kinase homolog capable of transforming 1-MNG enable strain JBH1 to grow on NG as the sole source of carbon and nitrogen. This rare ability makes JBH1 an excellent candidate for bioaugmentation or other strategies for bioremediation of NG contamination. The remaining questions about the assimilation of the carbon skeleton of NG and how the pathway evolved are under investigation.

ACKNOWLEDGMENTS

This work was supported by the DuPont Corporate Remediation Group. Additional support was provided by Defense Threat Reduction Agency/U.S. Army Research Office.

We thank Shirley Nishino, Marco Minoia, Zohre Kurt, Sara Craven, and Sachiyo Mukherji for helpful suggestions.

FOOTNOTES

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