Bacterial bioreporters for the detection of trace explosives: performance enhancement by DNA shuffling and random mutagenesis

Landmines and other explosive remnants of war pose a global humanitarian problem that claims numerous casualties long after the conflict has ended. As there are no acceptable methodologies for the remote discovery of such devices, current detection practices still require the risky presence of personnel in the minefield. We have recently described bacterial sensor strains capable of reporting the existence of 2,4-dinitrotoluene (DNT) vapors in the soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating a bioluminescent or a fluorescent signal. This may allow the identification of landmine location by remote imaging of an area over which the bacteria have been spread. In the study reported herein, we have improved the DNT-detection capabilities of these sensor strains by combining two DNT-responsive Escherichia coli gene promoters, yqjF and azoR, and subjecting them to three cycles of random mutagenesis by error-prone PCR, combined with segmentation and rearrangement ("DNA shuffling"). The activity of selected modified promoters was evaluated with the Aliivibrio fischeri and Photobacterium leiognathi luxCDABEG gene cassettes as the bioluminescent reporters, exhibiting a ten-fold background reduction that has led to a three-fold decrease in detection threshold. Signal intensity was further enhanced by modifying the ribosomal binding site of the yqjF gene promoter. The superior DNT detection capabilities on a solid matrix by the improved sensor strain were demonstrated. KEY POINTS: • Performance of microbial sensor strains for buried explosives was molecularly enhanced. • Manipulations included random mutagenesis, "DNA shuffling," and RBS reprogramming. • The re-engineered constructs exhibited superior detection of trace explosives.