Identification of 4-Amino-2-Nitrophenol as a novel inducer of phenotypic antibiotic resistance in Escherichia coli: roles of Lon protease and its substrate MarA

Abstract In prokaryotes, the energy-dependent protein degradation is controlled primarily by two ATP-dependent proteases, Lon and Clp. This study investigates the roles of the Lon protease in the metabolism of 2,4-dinitrophenol (2,4-DNP), a toxic industrial compound, in Escherichia coli (E. coli). During the study, an observation was made that the absence of Lon protease resulted in an enhanced conversion of yellow coloured 2,4-DNP to a reddish-brown product. This study aims to characterise the compound observed in the media with wild type (WT) and Δlon strains, understand the mechanisms of 2,4-DNP conversion and decipher the roles of Lon protease in the conversion of 2,4-DNP. UV-visible and LC-MS analyses revealed differences in the conversion products between the WT and Δlon strains. One of the substrates of Lon protease is MarA, a transcription factor. Growth studies with different mutants and trans-complemented strains demonstrated MarA-dependent conversion. The bathochromic shift of spectral peaks suggested a reduction process and possible involvement of nitroreductase enzymes. Indeed, the expression of two nitroreductases, nfsA and nfsB, increased with 2,4-DNP and was dependent on MarA. Importantly, the production of the reddish-brown product was lower in strains lacking nfsA or nfsB. Finally, LC-MS analysis identified one of the conversion products of 2,4-DNP to be 4-Amino-2-nitrophenol (4,2-ANP). Dose studies with purified 4,2-ANP demonstrated that it did not lower the growth of E. coli (unlike 2,4-DNP) but induced phenotypic antibiotic resistance (like 2,4-DNP). This study contributes to our understanding of biological treatment of nitroaromatics and may offer insights into environmental pollution mitigation strategies. Importance This study identifies the roles of Lon protease and its substrate MarA in inducing nitroreductases, NfsA and NfsB, in reducing toxic 2,4-DNP to less toxic 4,2-ANP, a novel inducer of phenotypic antibiotic resistance. This study contributes to understanding the biological treatment of nitroaromatics, offering insights into environmental pollution mitigation strategies and the development of efficient bioremediation techniques. Competing Interest Statement The authors have declared no competing interest.