The unchecked dispersal of antipersonnel landmines since the late 19th century has resulted in large areas contaminated with these explosive devices, creating a substantial worldwide humanitarian safety risk. The main obstacle to safe and effective landmine removal is the identification of their exact location, an activity that currently requires entry of personnel into the minefields; to date, there is no commercialized technology for an efficient stand-off detection of buried landmines. In this article, we describe the optimization of a microbial sensor strain, genetically engineered for the remote detection of 2,4,6-trinitrotoloune (TNT)-based mines. This bioreporter, designed to bioluminescence in response to minute concentrations of either TNT or 2,4-dinitotoluene (DNT), was immobilized in hydrogel beads and optimized for dispersion over the minefield. Following modifications of the hydrogel matrix in which the sensor bacteria are encapsulated, as well as their genetic reporting elements, these sensor bacteria sensitively detected buried 2,4-dinitrotoluene in laboratory experiments. Encapsulated in 1.5 mm 2% alginate beads containing 1% polyacrylic acid, they also detected the location of a real metallic antipersonnel landmine under field conditions. To the best of our knowledge, this is the first report demonstrating the detection of a buried landmine with a luminescent microbial bioreporter.

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The aromatic compound 2,4-dinitrotoluene (DNT), a common impurity in 2,4,6-trinitrotoluene (TNT) production, has been suggested as a tracer for the presence of TNT-based landmines due to its stability and high volatility. We have previously described an Escherichia coli bioreporter capable of detecting the presence of DNT vapors, harboring a fusion of the yqjF gene promoter to a reporter element. However, the DNT metabolite which is the direct inducer of yqjF, has not yet been identified, nor has the regulatory mechanism of the induction been clarified. We demonstrate here that the YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator of yqjF activation, as well as of a panel of additional E. coli genes. This group of genes share a common sequence motif in their promoters, which is suggested here as a putative YhaJ-box. In addition, we have linked YhaJ to the regulation of quinol-like compound degradation in the cell, and identified yhaK as playing a role in the degradation of DNT.

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli-based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter’s molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.

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