Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes.
Bottom Line: When strains bearing transcriptional fusions to reporters with an optical output (luxAB, GFP) were spread on soil spotted with nitrotoluenes, the signal triggered by promoter activation allowed localization of the target compounds on the soil surface.Our data provide a proof of concept that non-natural transcription factors evolved to respond to nitroaromatics can be engineered in soil bacteria and inoculated on a target site to pinpoint the presence of explosives.This approach thus opens new ways to tackle this gigantic humanitarian problem.
Affiliation: Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.Show MeSH
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Mentions: Pseudomonas putida Pu→lacZ cells carrying separately plasmids pCON916 (xylR+), pCON922 (xylR3+), pCON924 (xylR5+) and pURXAV17(xylRV17+) were subject to TNT induction experiments in liquid medium in the same conditions as those used for examining the response to 2,4 DNT, the results being shown in Fig. 6A. Although not as pronounced as in the case of 2,4 DNT, we could systematically record an increase of the β‐galactosidase levels in cells bearing xylR5 and xylRV17 which had been grown in cultures with TNT. In contrast, strains with the wild‐type xylR or the xylR3 variant did not react to the presence of the compound. In order to investigate the potential of this result for in situ visualization of TNT in soil, we passed plasmids pCON916 (xylR+), pCON922 (xylR3+), pCON924 (xylR5+) and pURXAV (xylRV17) to strain P. putida Pu→GFP and reproduced the experiments of Fig. 5 using TNT instead of 2,4 DNT as the test explosive residue. In contrast to the data of Fig. 6A, neither xylR5 nor xylRV17 brought about any significant fluorescent signal in colonies grown in the proximity of TNT (not shown). However, cells bearing the xylR3 mutant increased its fluorescent output quite above the basal level without inducer (Fig. 6B). That the response of the mutants to TNT depends on whether the assays are made on water‐saturating conditions (liquid cultures of Fig. 6A) or cells grown under matric stress (Fig. 6B) is intriguing. Although high‐purity TNT was used in the assays, we cannot altogether rule out that the XylR mutants detect small amounts of contaminating 2,4 DNT instead of sensing bona fide TNT. Should this be the case, the contaminant would be in a much lower concentration than the levels found in actual explosives and therefore the strain would still be useful as a biosensor. While this issue deserves some clarification, we argue that the A domain of the XylR protein is a suitable scaffold for developing whole‐cell bioindicators for a large variety of chemicals (Galvao and de Lorenzo, 2005b).
Affiliation: Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.