The Pseudomonas putida NfnB nitroreductase confers resistance to roxarsone

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 P. putida 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

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. Cells 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.

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 promoters 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 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.