There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one.We previously reported the isolation of Enterobacter cloacae PB2 on the basis of its ability to use nitrate esters such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate (GTN) as nitrogen sources. E. cloacae PB2 possesses a soluble PETN reductase capable of reductively liberating nitrite from nitrate esters with oxidation of NADPH (1) (Fig. 1). PETN reductase is a monomeric flavoprotein of M r 40 000. Recently, White et al. (20) reported the isolation of a strain of Agrobacterium radiobacter capable of growth with GTN as the sole nitrogen source and showed that cell extracts from this organism liberated nitrite from GTN and PETN with oxidation of NADH, suggesting the activity of a similar enzyme.Nitrate esters, though produced in large amounts for use as explosives and vasodilators (13), are rare in nature (7, 12), and multiply substituted nitrate esters are not known to occur naturally. The origin of enzymes apparently specialized for their breakdown is therefore of interest. To investigate this question, the structural gene encoding PETN reductase, designated onr (for organic nitrate reductase), was cloned using degenerate oligonucleotide probes.Cloning and sequence analysis of onr. The N-terminal sequence of PETN reductase purified from E. cloacae PB2 as previously described (1) was found to be SAEKLFTPLKV GAVTAPNRVFMAPLT. On the basis of E. cloacae typical codon usage, the following oligonucleotide probes were designed, based on residues 2 to 11 and 18 to 26, respectively:Southern blots using standard procedures (15) showed that both probes bound to the same region of PB2 genomic DNA. A 1,525-bp NcoI-ClaI genomic DNA fragment was cloned in pBluescript SKϩ (Stratagene) to give pONR1. Sequencing indicated the presence of an open reading frame beginning with codons matching the known N-terminal sequence of PETN reductase. The sequence of onr and deduced amino acid sequence of PETN reductase are shown in Fig. 2. The sequence predicts a protein of 364 residues with an M r of 39,358 excluding the N-terminal methionine, consistent with the M r of approximately 40,000 estimated for PETN reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1). A putative ribosome-binding site and transcription termination sequence were detected. No obvious 70 -like promoter sequence was present, but three regions between a putative upstream termination sequence and the ribosome-binding site have significant homology to Escherichia coli S -dependent promoters (21). The deduced amino acid sequence of PETN reductase was compared to sequences in protein and nucleic acid databases using the BLAST program of the GCG package (6). The most similar prote...

Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil.

This article provides a review of the published literature describing the use of biosensors and biologically-inspired systems for explosives detection. The review focusses on the use of antibodies, enzymes, biologically-inspired synthetic ligands and whole-cell biosensors, providing a flavour of the range of technology, formats and approaches that can be used to detect explosives using biological systems.