Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives

Abstract

DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an bioreporter strain engineered to detect traces of DNT and have demonstrated that the gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in , have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces is 2,4,5-trihydroxytoluene. We further show that cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells. The information provided in this article sheds new light both on the microbial biodegradability of nitroaromatic compounds and on the metabolic capabilities of By doing so, it also clarifies the pathway leading to the previously unexplained induction of the gene by 2,4-dinitrotoluene, an impurity that accompanies 2,4,6-trinitrotoluene (TNT)-based explosives. Our improved understanding of these processes will serve to molecularly enhance the performance of a previously described microbial bioreporter of buried landmines and other explosive devices, in which the gene promoter serves as the sensing element.

Detection of buried explosives with immobilized bacterial bioreporters

“…The optical response of these bioreporters to the presence of landmines in the soil below them would then be remotely imaged and analysed. Several microbial sensor strains have since been described (Looger et al, 2003;Altamirano et al, 2004;Radhika et al, 2007;Garmendia et al, 2008;Davidson et al, 2012;Yagur-Kroll et al, 2014;Shemer et al, 2015Shemer et al, , 2017Shemer et al, , 2018Shemer et al, , 2020) that emit a dose-dependent signal in the presence of the main explosive landmine constituent (Jenkins et al, 2001), 2,4,6-trinitrotoluene (TNT), and/or of its manufacturing by-product 2,4-dinitrotoluene (DNT). The latter compound, due to its relatively high volatility, is considered a useful 'signature chemical' for the presence of TNT-based landmines.…”

“…(2014) reported an Escherichia coli sensor strain, harbouring a plasmid‐borne fusion of the green fluorescent protein GFPmut2 to the E. coli yqjF gene promoter. The latter promoter was shown to be activated by both TNT and DNT (Yagur‐Kroll et al ., 2014), and directly induced by their degradation product 2,4,5‐trihydroxytoluene (Shemer et al ., 2018). The yqjF promoter was further optimized by directed evolution (Yagur‐Kroll et al ., 2015), and bacteria hosting the enhanced plasmid, encapsulated in Ca–alginate beads, successfully detected real antipersonnel landmines over which they were spread (Belkin et al ., 2017).…”

Enhancing DNT Detection by a Bacterial Bioreporter: Directed Evolution of the Transcriptional Activator YhaJ

“…Prominent among these reports is a description of E. coli -based DNT/TNT bioreporters harboring a genetic fusion between E. coli ’s endogenous yqjF gene promoter to either the green fluorescent protein gene GFPmut2 or to Photorhabdus luminescens bioluminescence luxCDABE genes ( Yagur-Kroll et al, 2014 , 2015 ). While the fluorescent variant has been instrumental in demonstrating the standoff detection of real antipersonnel landmines ( Belkin et al, 2017 ), recent efforts have concentrated on molecularly enhancing the performance of the bioluminescent variants ( Shemer et al, 2020 ; 2021 ; Shpigel et al, 2021 ), in parallel to unravelling the DNT degradation pathway ( Shemer et al, 2018 ) and the yqjF regulatory mechanism in E. coli ( Palevsky et al, 2016 ). The latter study has pointed at YhaJ, a member of the LysR type family of transcriptional regulators, as a positive regulator of yqjF activation, linked to aromatic compounds degradation.…”

Biotransformation of 2,4‐dinitrotoluene in a phototrophic co‐culture of engineered Synechococcus elongatus and Pseudomonas putida

“…Reduction of the nitro group of 2,4-DNT creates highly reactive nitroso-intermediates and hydroxylamino-derivatives, which are also toxic due to their capacity to bind a range of biomolecules, frequently causing cellular damage by forming DNA and protein adducts (Spain, 1995;Achtnich et al, 1999;Padda et al, 2003). The toxic derivatives formed by the reductive pathway are not fully mineralized by most bacteria and also have long persistence times in natural environments (Achtnich et al, 1999;Shemer et al, 2018). A separate pathway that initiates with 2,4-DNT oxidation has been identified in some prokaryotes, including Burkholderia sp.…”

“…The non-specific reductive pathway is thought to involve reduction of 2,4-DNT by common electron carriers such as flavinor iron center-containing electron donors (e.g. nitroreductases; French et al, 2001;Shemer et al, 2018). Reduction of the nitro group of 2,4-DNT creates highly reactive nitroso-intermediates and hydroxylamino-derivatives, which are also toxic due to their capacity to bind a range of biomolecules, frequently causing cellular damage by forming DNA and protein adducts (Spain, 1995;Achtnich et al, 1999;Padda et al, 2003).…”