The Pseudomonas putida NfnB nitroreductase confers resistance to roxarsone

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.