The Pseudomonas putida NfnB nitroreductase confers resistance to roxarsone

Abstract

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox) has been used for decades as an antimicrobial growth promoter for poultry and swine. Roxarsone is excreted in chicken manure unchanged and can be microbially transformed into a variety of arsenic-containing compounds such as 3-amino-4-hydroxyphenylarsonic acid (HAPA(V)) that contaminate the environment and present a potential health hazard. To cope with arsenic toxicity, nearly every prokaryote has an ars (arsenic resistance) operon, some of which confer resistance to roxarsone. Pseudomonas putida KT2440 is a robust environmental isolate capable of metabolizing many aromatic compounds and is used as a model organism for biodegradation of aromatic compounds. Here we report that P. putida KT2440 (ΔΔars) in which the two ars operons had been deleted retains resistance to highly toxic trivalent Rox(III), the likely active form of roxarsone. In this study, a genomic library constructed from P. putida KT2440 (ΔΔars) was used to screen for resistance to Rox(III) in Escherichia coli. One gene, termed, PpnfnB, was identified that encodes a putative 6,7-dihydropteridine reductase. Cell expressing PpnfnB reduce the nitro group of Rox(III), and purified NfnB catalyzes FMN-NADPH-dependent nitroreduction of Rox(III) to less toxic HAPA(III). This identifies a key step in the breakdown of synthetic aromatic arsenicals.

Graphical Abstract

1. Introduction

Roxarsone and related synthetic aromatic arsenicals such as nitarsone (4-nitrophenylarsonic acid, Nit) and p-arsanilic acid (4-aminophenylarsonic acid, pASA) are organoarsenical compounds that have been widely used for decades in poultry and swine as feed additives to prevent coccidiosis infections and enhance growth (Nachman et al., 2013). Little ingested roxarsone is retained in the chicken tissue and most is excreted unchanged in the chicken feces (Mafla et al., 2015). Unmetabolized roxarsone is ultimately released into the environment with the animal manure, which is a commonly used as fertilizer for agricultural production. Until recently in the United States approximately 1000 tons of roxarsone were released into environments annually from manure fertilizer (Han et al., 2017). The use of aromatic arsenical growth promotors is no longer permitted in either the United States (https://www.fda.gov/animalveterinary/product-safety-information/arsenic-based-animaldrugs-and-poultry) or the European Union and was recently banned in China (Tang et al., 2019). However, roxarsone is still used as an animal feed supplement in developing countries such as Brazil and India, and compliance in countries where it is banned has been questioned (Hu et al., 2013; Huang et al., 2019; Yin et al., 2018). The U.S. Environmental Protection Agency's (EPA’s) list of priority pollutants for environmental remediation designates nitroaromatic compounds such as Rox as hazardous to human health (Ju and Parales, 2010). Although pentavalent organoarsenicals such as roxarsone and nitarsone have low toxicity, they are degraded into more toxic metabolites after the manure is applied as fertilizer or composted (Garbarino et al., 2003). In an oxic environment, a common step in microbial biotransformation of pentavalent Rox(V) is reduction of the nitro group to form the amine 3-amino-4-hydroxyphenylarsonic acid (HAPA(V)), which is relatively more stable but is eventually degraded into inorganic As(V) and As(III), with many identified intermediate species such as dimethylarsenate (DMAs(V)), methylarsenate (MAs(V)), HAPA(V), 3-acetamido-4-hydroxyphenylarsonic acid (N-AHAA) and smaller amounts of other organoarsenicals (Fisher et al., 2008; Yang et al., 2016; Yao et al., 2019). Recently, additional organoarsenical degradative products have been isolated and identified during biodegradation by both aerobes and anaerobes, including the more toxic and mobile species trivalent HAPA(III) and Rox(III) (Frensemeier et al., 2017). A number of environmental microbial isolates, both anaerobes and aerobes, show the ability to biotransform roxarsone, including the obligate aerobe Alkaliphilus oremlandii OhILAs (Fisher et al., 2008), the facultative anaerobes Shewanella oneidensis MR-1 (Chen et al., 2018b) and S. putrefaciens 200 (Chen and Rosen, 2016) and obligate anaerobic Clostridia species. Under anaerobic methanogenic and sulfate-reducing conditions, the nitro group of Rox(V) could rapidly be reduced and form the amine HAPA(V), which is slowly but finally broken down to form inorganic As(III) and As(V) (Cortinas et al., 2006). Recently Enterobacter sp. CZ-1 isolated from an arsenic-contaminated paddy soil was shown to not only reduce the nitro group of Rox(V) to HAPA(V), but then to acetylate the amino group to generate N-acetyl-4-hydroxy-m-arsanilic acid (Huang et al., 2019). S. putrefaciens 200 can also transform trivalent Rox(III) and Nit(III) into HAPA(III) and p-ASA(III), respectively. S. putrefaciens 200 has an arsenic gene island that includes three genes, arsEFG, that have synergistic interaction and catalyze independent reduction of the nitro group and arsenic atom coupled to efflux of the reduced trivalent aminoaromatic arsenicals (Chen et al., 2019b). The legume symbiont Sinorhizobium meliloti can reduce Rox(V) to trivalent HAPA(III) in two independent and sequential reductions of the nitro group and arsenic atom (Yan et al., 2019). In that study the S. meliloti Rm1021 enzyme MdaB was shown to be an FAD-NADPH-dependent nitroreductase that catalyzes nitroreduction of pentavalent roxarsone. These results demonstrate that there are multiple ways to transform either pentavalent or trivalent roxarsone to either pentavalent or trivalent HAPA under either anaerobic or aerobic conditions and suggest that more genes/enzymes that carry out these reactions likely exist.

To search for new genes/enzymes involved in roxarsone detoxification/degradation, we investigated the ability of P. putida KT2440 to transform roxarsone. P. putida is widely distributed soil saprophytic bacterium that efficiently colonizes plant roots. It shows a remarkable adaptability to diverse environments including soil contaminated with multiple heavy metals and nitro-aromatic compounds (Fernandez et al., 2013). This soil microorganism has been exploited extensively and effectively as an experimental model for the biodegradation of aromatic compounds such as such as benzene and toluene (Molina-Santiago et al., 2016). P. putida KT2440 has two ars operons for highly arsenic tolerance, ars1 and ars2, both of which have arsRBCH genes (Paez-Espino et al., 2015), and ars1 has three other genes of unknown function. Deletion of both operons (P. putida KT2440 (ΔΔars)) resulted in arsenic hypersensitivity. In this study, we show that both wild type P. putida wild type and the double ars operon deleted strain (ΔΔars) are resistance to toxic Rox(III) and transform Rox(III) to HAPA(III) by reduction of the nitro group to an amine. We constructed a genomic DNA library from P. putida ΔΔars. In a screen for resistance to Rox(III), we identified a gene, PpnfnB, which encodes a putative FMN-NADPH-dependent nitroreductase, that confers resistance to Rox(III). Cells of E. coli expressing PpnfnB reduced the nitro group of Rox(III) to an amine, forming less toxic HAPA(III). Purified NfnB reduces the nitro groups of both Rox(III) and Nit(III) to form the corresponding aromatic amines HAPA(III) and p-ASA(III), respectively. These results demonstrate that NfnB biotransformation confers resistance to environmental aromatic arsenicals. The extensive phylogenetic distribution of nfnB genes indicates how roxarsone is widely biotransformed into HAPA by microorganisms in both anaerobic and aerobic environments.

2. Material and methods

2.1. Chemicals

All chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich. Roxarsone was obtained from ThermoFisher Acros Organics Division (Waltham, MA) and Chem Service (West Chester, PA). Nit(V), PhAs(III)), HAPA(V) and p-ASA were purchased from Sigma-Aldrich (St Louis, MO). Pentavalent arsenicals were reduced as described (Reay and Asher, 1977). The reduced products were not thiolated, as determined by simultaneous As and S analysis by high pressure liquid chromatography (HPLC) coupled with inductively coupled mass spectroscopy (ICP-MS) (ELAN DRC-e; Perkin-Elmer, Waltham, MA) (Qin et al., 2006).

2.2. Strains, medium and growth conditions

E. coli Stellar™(Clontech Laboratories, Mountain View, CA) (F, endA1, supE44, thi-1, recA1, relA1, gyrA96 phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF)U169, Δ(mrrhsdRMS-mcrBC), ΔmcrA, λ-) was used for plasmid DNA construction and replication. E. coli AW3110 (Δars::cam F-IN(rrn-rrnE) (Qin et al., 2006), which is hypersensitive to As(III), was used for Rox(III) resistance complementation studies. For most experiments, cultures of E. coli were grown aerobically at either 30 °C or 37 °C in either lysogeny broth (LB) medium or M9 minimal medium with 0.2% glucose (Sambrook et al., 1989), as noted, supplemented with 125 μg mL⁻¹ ampicillin, 50 μg mL⁻¹ kanamycin or 34 μg mL⁻¹ chloramphenicol, as required (Chong, 2001). P. putida cells were grown aerobically at 30 °C in either LB or M9 medium with citrate (2 g/L) as the sole carbon source and 20 μg mL⁻¹ uracil (Paez-Espino et al., 2015).

2.3. Cloning a gene for Rox(III) resistance

Total DNA was isolated from a 50 mL culture of P. putida KT2440 ΔΔars (Paez-Espino et al., 2015). The extracted DNA was partially digested with PstI (New England BioLabs) and ethanol-precipitated. The pelleted DNA was suspended in water and layered on a discontinuous sucrose gradient (10%, 20%, 30%, and 40%, wt/vol) containing 50 mM Tris, pH 8.0, 0.1 M EDTA, and 0.1 M NaCl, and centrifuged at 210,000 × g for 5 h at 17 °C. After centrifugation, the solution was carefully removed from the top of the layer and separated into fractions. Fractions containing DNA fragments of more than 2 kbp were combined and concentrated by ethanol precipitation. The resulting DNA was ligated to Pstl-digested plasmid pUC118 (Takara Bio), and the ligation mixture was transformed into E. coli strain TOP10 (Invitrogen) using a MicroPulser (Bio-Rad). The transformants were spread on M9 agar plates containing 100 μg/mL ampicillin, citrate (2 g/L), 20 μg mL⁻¹ uracil, and 2 μM Rox(III) and incubated at 30 °C until colonies formed. The plates were replicated three times onto plates containing 2 μM Rox(III) and incubated at 30 °C until colonies formed. Clones were cultured in LB medium supplemented with 100 μg/mL ampicillin at 30 °C overnight, transferred to M9 minimal medium supplied with citrate, uracil and 2 μM Rox(III) and incubated at 30 °C to confirm Rox(III) resistance. Plasmid DNA from Rox(III) resistant clones were sequenced using Sequetech DNA Sequencing Services (Mountain View, CA).

2.4. Plasmid construction

The DNA sequence of the resistant clone had only a single full-length gene and two partial genes. The full-length gene was annotated as a putative 6,7-dihydropteridine reductase that we termed PpnfnB. The gene was subcloned into plasmid pET28 (Novagen) by introducing an Ncol site at the 5-end and an Xhol site at the 3-end. The forward and reverse primers were 5’GGAGTTGTCATATGGATACCGTATCGCTGG-3’ and 5’AAAGCGGCCCCAAAAACTCTCGAGGAAGGT-3’ respectively. The amplified product was gel purified, digested with Ncol and Xhol, and inserted into the Ncol-Xhol sites of pET28a (+) vector, in frame with the C-terminal six-histidine-residue tag. The construct was confirmed by DNA sequencing.

2.5. Metalloid resistance assays

For metalloid resistance assays in liquid medium, competent cells of AW3110 were transformed with the plasmid bearing the PpnfnB gene or vector plasmid and induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cultures of P. putida or E. coli were grown in LB medium at 30 °C to an A_600nm of 2.0. Over night cultures were diluted 100-fold in fresh M9 medium with all required additives. Cells were cultured for 16 h with shaking at 30 °C with organoarsenicals at the indicated concentrations.

2.6. Assay of reduction of aromatic arsenicals

Hydroxynitro aromatic compounds have a strong absorbance at 410 nm (Zeyer and Kocher, 1988). Both Rox(III) and Rox(V) are yellow in solution. Reduction of the nitro substituent to an amine can be observed visibly by loss of the yellow color and spectrophometrically from the decrease in absorption at 410 nm. HAPA, p-ASA, and nitarsone do not have hydroxynitro substituents and do not absorb at 410 nm. E. coli AW3110 cells with the PpnfnB gene were cultured aerobically with shaking in LB medium overnight at 37 °C. The cells were washed once and suspended in M9 medium without glucose at a cell density of A_600nm =4.0.Rox(III)or p-nitrophenol was then added at the indicated concentrations to the cell suspensions, which were incubated at 30 °C with shaking for 4 h. The nitroreductase activity of cells with the PpnfnB gene was quantified by loss of absorbance at 410 nm that was measured with a Synergy H4 Hybrid Multi-Mode microplate reader.

A second assay for nitroreduction employed analysis by HPLC-ICP-MS. Cells of P. putida or E. coli AW3110 expressing the PpnfnB gene were cultured aerobically with shaking in LB medium overnight at 30 °C. The cells were washed and suspended in M9 medium at an A_600nm=3.0. Rox(III) was added at 4 μM, final concentration, to the cell suspensions, which were incubated at 30 °C with shaking for 4 h. Soluble arsenicals were speciated by HPLC (Series 2000; Perkin-Elmer, Waltham, MA) coupled to ICP-MS (ELAN DRC-e; Perkin-Elmer) using either a Jupiter® 5 μm C18 300 Å reverse-phase column (250 mm × 4.6 mm; Phenomenex, Torrance, CA) eluted isocratically with a mobile phase consisting of 3 mM malonic acid, 5 mM tetrabutylammonium hydroxide, and 5% methanol (v/v), pH 5.6, with a flow rate of 1 mL min-1 at 25 °C or with an Inertsil® 5 μm C4 150 Å reverse-phase column (150 mm × 2.1 mm; GL Sciences, Torrance, CA) eluted with 15% acetonitrile (v/v), 15% ethanol (v/v), 80% water (v/v), pH 1.5, with a flow rate of 0.8 mL min-1 at 60 °C. Some arsenic was not recovered in these experiments because variable amounts were bound to cellular constituents.

2.7. NfnB purification and assay

E. coli TOP10 cells (Life Technologies) bearing PpnfnB in plasmid pET28a (+) were grown in LB medium containing 50 μg mL⁻¹ kanamycin with shaking at 37 °C. At an A_600nm of 0.6, 0.3 mM IPTG was added as an inducer, and the culture was grown for an additional 4 h at 37 °C. The cells were harvested and suspended in 5 mL per gram of wet cells in buffer A (50 mM MOPS (4-morpholinepropanesulfonic acid), 20 mM imidazole, 0.5 M NaCl, 10 mM 2-mercaptoethanol and 20% glycerol (vol/vol), pH 7.5). The cells were broken by a single passage through a French pressure cell at 20,000 psi. The protease inhibitor diisopropyl fluorophosphate was immediately added at 2.5 μL per gram wet cells. Membranes and unbroken cells were removed by centrifugation at 150,000 × g for 1 h, and the supernatant solution was loaded onto a Ni²⁺-nitrilotriacetic acid column (Qiagen, Valencia, CA) at a flow rate of 0.5 mL min⁻¹. The column was washed with more than 20 column volumes of buffer A. NfnB was eluted with buffer A containing 0.2 M imidazole, and purity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) (Laemmli). The protein concentration of purified NfnB was estimated its calculated extinction coefficient at A_280nm (ε = 20,460M⁻¹ cm⁻¹). NfnB-containing fractions were divided into portions, rapidly frozen, and stored at −80 °C until use.

Flavin mononucleotide (FMN) in purified NfnB was quantified by absorption at 450 nm using a molar extinction coefficient of 12020 M⁻¹·cm⁻¹. Reduction of FMN by NfnB was assayed in 25 mM Bis-Tris propane buffer (pH 7.0) containing 1 mM EDTA and 0.1 mg mL⁻¹ bovine serum albumin (BSA) at 37 °C. NADPH (0.2 mM) was incubated with 1 μM NfnB and/or 25 μM FMN, and the oxidation of NADPH was monitored from the decrease in absorbance at 340 nm (ε = 6220 M⁻¹·cm⁻¹).

The nitroreductase activity of NfnB was examined in vitro using purified protein. Rox(V), Rox(III) Nit(V) or Nit(III) (4 μM) was incubated at 37 °C in the presence or absence of 1 μM NfnB in a reaction solution (25 mM Tris, 25 mM Bis-Tris propane (pH 7.0), 1 mM EDTA, 0.1 mg mL⁻¹ BSA, 0.2 mM NADPH and 25 μM FMN, 5 mM glutathione (GSH) and 0.5 mM tris-(2-carboxyethyl)phosphine (TCEP)). Reactions were collected at the indicated time points, and the arsenic species were analyzed by HPLC-ICP-MS, as described above.

3. Results and discussion

3.1. P. putida is resistant to Rox(III)

The relative biological availability and toxicological effects of arsenic depend primarily on its dose, chemical nature and oxidation state (Hughes et al., 2011). Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite (Chen et al., 2014). The effect of organoarsenicals on P. putida KT2440 was examined. A strain with both ars operons deleted (ΔΔars) remains resistant to highly toxic Rox(III) (Fig. 1). Wild type P. putida KT2440 is resistant to MAs(III), PhAs(III), Nit(III) and p-ASA(III), but the double deletion strain is sensitive to MAs(III), PhAs(III), Nit(III) and p-ASA(III) because of deletion of the arsH genes (Chen et al., 2015). Other bacteria have genes such as arsI, arsP, arsK and arsEFG that confer various levels of resistance to Rox(III), but these genes are not present in the P. putida genome (Chen et al., 2019a). Resistance to Rox(III) in the double ars deletion strain P. putida ΔΔars points to alternative resistance mechanisms for Rox(III) resistance in this bacterium.

Fig. 1. P. putida ΔΔars is resistant to Rox (III).

Overnight cultures of P. putida ΔΔars were diluted 100-fold into fresh M9 medium containing growth additives and various arsenic compounds, as indicated. Growth was measured after 16h at 30°C. Data are the mean ±SE (n=3).

3.2. Reduction of Rox(III) by P. putida

Both P. putida wild type and ΔΔars strains have the Rox(III) resistance phenotype. We previously showed that the soil bacterium S. meliloti Rm1021 can reduce the nitro group of Rox(V) to HAPA(V), and sequentially the pentavalent arsenic was reduced to HAPA(III) (Yan et al., 2019). However, wild type P. putida KT2440 does not transform the aromatic organoarsenicals Rox(V) and Nit(V), perhaps due to its poor uptake. In contrast, both the wild type and ars deletion strains reduce Rox(III) to HAPA(III) (Fig. 2A and Fig. S1). The wild type strain transformed Rox(III) to both HAPA(III) and HAPA(V), while the deletion strain produced less HAPA(V), probably because it lacks the arsH gene that encodes an organoarsenical oxidase (Chen et al., 2015). HAPA(III) and HAPA(V) appear to be end products, with no further degradation detected in P. putida. Compared to Rox(III), HAPA(III) is much less toxic in mammalian cells (Peng et al., 2017), and we previously showed that E. coli is resistant to HAPA(III) even at 10 μM (Chen et al., 2019b). Here we show that HAPA(III) is much less toxic in P. putida as well (Fig. 1). It is therefore reasonable to conclude that nitroreduction of Rox(III) to HAPA(III) is a detoxification pathway.

Fig. 2.

Rox transformation by P. putida ΔΔars and E. coli. Transformation of Rox by P. putida KT2440 wild type and ΔΔars (A) and E. coli with cloned P. putida genes (B). Arsenic species were assayed by HPLC-ICP-MS with a C18 reverse column, as described under Materials and Methods.

3.3. Identification of a gene for Rox(III) resistance

We screened for genes from the P. putida ΔΔars deletion strain that could confer Rox(III) resistance in E. coli. One resistant isolate reduced Rox(III) into HAPA(III). The peak of HAPA(III) was shifted to the position of HAPA(V) when oxidized by H₂O₂, confirming that the product is HAPA(III) (Fig. 2B). The sequence of the 2 kilobase insert was examined by a BLAST search of P. putida genomic DNA, which identified three putative genes (Fig. S2A). The first was annotated as a LysR transcriptional repressor. The second was annotated as PpnfnB, encoding a putative 6,7-dihydropteridine reductase. The third encodes a 159-residue conserved protein of unknown function. However, only the second gene was full length, and the other two were only partial genes, indicating that PpnfnB is responsible for Rox(III) resistance.

3.4. PpnfnB encodes a nitroaromatic arsenic nitroreductase

NfnB (NP_744580.1) is not related to other enzymes known to reduce Rox(III). For example, with only 23% similarity to S. meliloti 1021 SmMdaB (WP_013844866.1), which reduces the nitro group of Rox(V) to HAPA(V). This suggests that NfnB is a novel nitroaromatic arsenic nitroreductase that reduces Rox(III) and confers resistance in E. coli strain AW3110 (Δars) (Carlin et al., 1995) (Fig. S2B). E. coli strain AW3110 expressing PpnfnB did not confer resistance to As(III), MAs(III) or Nit(III) (Fig. 3A). In solution both Rox(V) and Rox(III) are yellow, while their amine derivatives HAPA(V) and HAPA(III) are colorless. Therefore, biotransformation by cells of E. coli AW3110 expressing the PpnfnB gene can be easily visualized by eye and quantified by the decrease in absorption at 410 nm. After 4 h of incubation, absorbance of Rox(III), which is directly proportional to its concentration, decreased in cells expressing PpnfnB (Fig. S3A and Fig. 3B). Cells with vector alone did not reduce Rox(III). These results indicate that Rox(III) is reduced by NfnB. Purified NfnB also reduces yellow p-nitrophenol to colorless 4-aminophenol, which indicates that NfnB is a nonspecific nitroreductase and did not evolve to confer Rox(III) resistance. This may explain why the PpnfnB gene is not in an ars operon, which contains genes specifically associated with arsenic biotransformations, transport and resistance (Mukhopadhyay et al., 2002). The chemical nature of the reduction product was further confirmed by HPLC-ICP-MS analysis (Fig. 3C). When cells expressing PpnfnB were incubated with 4 μM Rox(III) for 12 h, the Rox(III) peak decreased, and a new peak appeared at the elution position of both As(III) and HAPA(III), which co-elute and cannot be separated with a C4 reverse-phase column. As(III) and HAPA(III) can be separated on a C18 column. Using this column, the product eluted at the position of HAPA(III). The peak of HAPA(III) was shifted to the position of HAPA(V) when treated with H₂O₂, confirming the identification of the product as HAPA(III). Similar results were obtained with Nit(III) (Fig. S3B and S3C). Cells expressing the PpnfnB gene reduced Nit(III) to p-ASA(III) were not resistant to Nit(III), consistent with the observation that p-ASA(III) is more toxic than HAPA(III).

Fig. 3.

Resistance to Rox(III) is the resul t of reduct ion of the ni tro group. (A), PpnfnB confers E. coli resistance to Rox(III). (B), Reduction of nitro group of Rox(III) and p-nitrophenol monitored by the change in A_410nm. Rox(III) and p-nitrophenol at the indicated concentrations was added to cells of E. coli AW3110 expressing PpnfnB, and reduction of the nitro group was assayed from the decrease in absorbance at 410 nm. (C), Reduction and speciation of aromatic arsenicals. Nitroreduction was assayed in cells of E. coli AW3110 carrying either vector plasmid or PpnfnB. Arsenicals were speciated by HPLC using a C18 reverse-phase column. The amount of arsenic in each assay was estimated by ICP-MS.

3.5. NfnB is a nitroreductase

NfnB is predicted to be an NAD(P)H-dependent nitroreductase (NR). This class of enzymes usually bind with flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as a prosthetic group and generate the hydroxyamino or the amino derivative as the end product (Rau and Stolz, 2003; Roldán et al., 2008). Purified NfnB is yellow and exhibits an absorption spectrum with λ_max at 450 nm. The FMN occupancy of the purified protein was only 59% (Fig. S4A), and supplementation with exogenous FMN in addition to NADPH was required for optimal FMN reductase activity (Fig. S4B). NfnB nitroreductase activity with Rox(V) as substrate was stimulated in the present of a reductant such as GSH or TCEP for (Fig. 4A). NfnB also reduces Nit(V) to p-ASA(V) (Fig. 4B). These results show that NfnB can reduce both pentavalent and trivalent aromatic nitroarsenicals. The difference between the in vivo and in vitro results are probably due to lack of uptake of the pentavalent compounds by E. coli. According to their sensitivity to oxygen, bacterial NRs are classified as oxygen-insensitive (type 1) or oxygen-sensitive (type 2) enzymes (Peterson et al., 1979). NfnB is not oxygen sensitive and is therefore a type I NR. Our results clearly demonstrate that NfnB is a type I nitroaromatic reductase with broad substrate specificity that can reduce the nitro group of Rox(V)/(III), Nit(V)/(III) and p-nitrophenol.

Fig. 4.

Nitroreductase activity of purified PpNfnB. Nitroreduction of either (A) Rox(V) or (B) Nit(V) by purified PpNfnB was assayed as described under Materials and Methods. Nitroreduction of Rox(V) by PpNfnB was carried out with 5 mM GSH and 0.5 mM TCEP and analyzed by HPLC-ICP-MS to speciate the aromatic arsenical substrates and products.

Although no longer used in some countries, roxarsone continues to be used in other countries in poultry feed to kill intestinal parasites and promote growth. In addition, when chicken manure is used as fertilizer, roxarsone has detrimental effects on microbial community diversity and metabolic activity in soil (Jiang et al., 2013). Roxarsone and its metabolite HAPA(V) also changes the diversity and represses the bacterial genera in the soil microbial community responsible for nitrogen removal, such as nitrification and denitrification, and the coupling of nitrification and denitrification may have an important role in stimulating nitrate removal efficiency and phosphorus release in agroecosystems (Chen et al., 2018a). Roxarsone also inhibits the acetoclastic and hydrogenotrophic utilizing methanogenic microorganisms. The degradation of organic matter by microbial is potential important and methanogenesis is considered to be the final step in many anaerobic environments, including sediments, anoxic groundwater (Sierra-Alvarez et al., 2010). These considerations indicate that the continued use of roxarsone has serious environmental consequences.

4. Conclusion

Nitroaromatic compounds such as roxarsone can be biotransformed by microorganism into more toxic compounds that leach into surface waters and contaminate the environment. While ars operons evolved for arsenic detoxification, our results clearly show P. putida still confers resistance to Rox(III) even after deletion of its two ars operons by reduction of the nitro group to an amine. The PpnfnB gene was shown to confers Rox(III) resistance in E. coli and encodes an FMN-NADPH-dependent nitroreductase. Purified NfnB catalyzes nitroreduction of both Rox(V) and Rox(III), as well as Nit(V) and p-nitrophenol. Our results indicate that nitroreductases play key roles in the degradation and detoxification of aromatic arsenicals. The complete pathway of roxarsone degradation remains to be determined, which is why identification of specific genes and enzymes involved in its biotransformations is of significant environmental relevance.

Highlights.

• A Pseudomonas putida strain with the ars operons deleted strain is resistant to highly toxic trivalent roxarsone (Rox(III))

• Both wild type P. putida wild type and the ars deletion strains reduce Rox(III) to HAPA(III).

• The PpnfnB gene from a genomic library of the P. putida ars operons deleted strain was identified by selection for Rox(III) resistance in E. coli.

• The NfnB enzyme catalyzes FMN-NADPH-dependent nitroreduction, reducing Rox(III) to less toxic HAPA(III).

• Niitroreduction is an alternative pathway for resistance to roxarsone in addition to resistance determinants found in ars operons.

Abbreviations

Rox(V)

roxarsone (3-nitro-4 hydroxypheynylarsonic acid)

Rox(III)

roxarsone with trivalent As(III)

HAPA(V)

3-amino4-hydroxyphenylarsonic acid

HAPA(III)

HAPA(V) with trivalent As(III)

Nit(V)

nitarsone, 4-nitrophenylarsonic acid

Nit(III)

Nit with trivalent As(III)

p-ASA(V)

p-arsinilic acid (4-aminopheylarsonic acid, atoxyl)

pASA(III)

pASA with trivalent As(III)

MAs(IIII)

methylarsenite

MAs(V)

methylarsenate

DMAs(V)

dimethylarsenate

PhAs(III)

phenylarsenite

HPLC

high pressure liquid chromatography

ICP-MS

inductively coupled plasma mass spectroscopy