Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes

Abstract

Although different biological approaches for detection of anti-personnel mines and other unexploded ordnance (UXO) have been entertained, none of them has been rigorously documented thus far in the scientific literature. The industrial 2,4,6 trinitrotoluene (TNT) habitually employed in the manufacturing of mines is at all times tainted with a small but significant proportion of the more volatile 2,4 dinitrotoluene (2,4 DNT) and other nitroaromatic compounds. By using mutation-prone PCR and DNA sequence shuffling we have evolved in vitro and selected in vivo variants of the effector recognition domain of the toluene-responsive XylR regulator of the soil bacterium Pseudomonas putida that responds to mono-, bi- and trinitro substituted toluenes. Re-introduction of such variants in P. putida settled the transcriptional activity of the cognate promoters (Po and Pu) as a function of the presence of nitrotoluenes in the medium. 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.

Figure 1

Strategies for experimental evolution of the XylR protein.
A. Domain organization of XylR. Relevant portions of the protein include the signal reception (effector binding) N‐terminal A domain (amino acid residues 1–211), the B linker (211–233), the central C module involved in NTP binding and RNAP‐σ⁵⁴ activation (233–472), and the D domain at the C‐terminus, with a helix–turn–helix (HTH) motif for DNA binding (514–556).
B. Shuffling between the DNA sequences of the A domains of XylR and DmpR. The procedure (Garmendia et al., 2001) implies the generation and mix of DNA segments covering the sequences of interest and their rescue in pCON918, a broad‐host‐range vector designed for cloning the products of shuffling the A domain sequences of the NdeI–SnaBI fragments. The resulting plasmids are then passed to a Po→km/Po→sacB P. putida strain for selection of clones able to activate Po in the presence of Km and 2,4 DNT, and counterselection of constitutive xylR mutants in plates with sucrose.
C. Mutagenic PCR. Following error‐prone amplification of the DNA sequence of the xylR A domain, the resulting PCR products were prepared as an EcoRI–AvrII fragment and cloned in the corresponding sites of plasmid pURXAv, thereby reconstituting full‐length xylR. The ligation pool is then passed to P. putida TEC3 that bears chromosomal Pu→pyrF‐lacZ and Pu→km transcriptional fusions. This allowed selection of the clones responsive to 2,4 DNT by means of the conditionally expressed Km resistance and pyrF.
D. The outcome of either of these two procedures is the isolation of xylR variants encoding A domains that respond to 2,4 DNT.

Figure 2

Characterization of XylR variants responsive to 2,4 DNT.
A. Localization of the amino acid changes found in individual variants of the A and B XylR domain that cause sensitivity to 2,4 DNT.
B. Response of XylR mutants to 2,4 DNT. Cultures of the Pu → lacZ strain P. putida SF05 carrying plasmids encoding each of the xylR variants indicated were grown in LB medium to an A₆₀₀ ~ 1.2 and then added with 2 mM 2,4 DNT. Following a further 3 h incubation, accumulation of β‐galactosidase was measured as described in Experimental procedures. Values given represent the average of at least three independent experiments, each of which was conducted in duplicate samples, with deviations being less than 15%. ind, inducer.
C. Response of xylR variants to the natural XylR inducer toluene. Experiments were made as before, excepting that the cultures were exposed for 3 h to saturating vapours of the volatile inducer in an airtight flask.
D. Dose–response patterns of XylR, XylR3 and XylR5 to varying concentrations of 2,4 DNT. Induction conditions were identical with those described above excepting for the different concentrations of the inducer.

Figure 3

2,4 Dinitrotoluene‐dependent light emission by P. putida Po→luxAB bearing xylR variants.
A. 2,4 Dinitrotoluene‐dependent luminiscence test of P. putida Po→luxAB transformed with either pCON16 (xylR⁺) or pCON924 (xylR5⁺). Strains were streaked out on plates with or without 2 mM DNT and the light recorded on an X‐ray film. Note the excellent signal to noise ratio caused by xylR5 under these conditions.
B. Light emission in response to high 2,4 DNT concentrations. An agar LB plate was overlaid with a suspension of approximately 10⁶ colony‐forming units (cfu) of the same strains, on the centre of which an approximately 1 mg crystal of 2,4 DNT was deposited in the point indicated with the arrow.
C. The X‐ray film record of such an assay, in which a strong light emission concentrates in the close proximity of the inducer in contact with P. putida Po→luxAB (pCON924xylR5) cells. Similar results were observed with the equivalent strain bearing xylR3.

Figure 4

Emission of green fluorescence by P. putida Pu→GFP bearing xylR variants. pCON916 (xylR⁺), pCON922 (xylR3⁺) and pCON924 (xylR5⁺) encoding various xylR alleles were passed to strain P. putida Pu→GFP (Table 1) and streaked out on agar plates amended with 2 mM of either 2,4 DNT or the natural XylR effector 3‐methylbenzylalcohol (3MBA). After overnight growth, plates were illuminated either with white visible light (vis) or with blue light (BL) for exciting the fluorescence of GFP, as indicated in each case.

Figure 5

Detecting 2,4 DNT in soil‐agar microcosms spread with reporter bacteria. Pseudomonas putida Pu→GFP cells transformed with pCON924 (xylR5⁺) were unevenly sprinkled on soil plates immobilized with soft agar and either exposed to 2,4 DNT vapours or blotted with small dots of the solid compound. As a controls, the P. putida Pu→GFP strain transformed with pCON916 encoding wild‐type xylR was tested under the same conditions. Note a vigorous fluorescent signal in the parts of the plate where bacterial growth has direct contact with the inducer.

Figure 6

Responses of 2,4 DNT‐sensitive variants of XylR to 2,4,6 trinitrotoluene (TNT).
A. Liquid medium assays. Pu→lacZ strain P. putida SF05 transformed with plasmids encoding each of the xylR variants indicated were grown in LB medium and added with 1 mM TNT in the same conditions explained in the legend to Fig. 2B. β‐Galactosidase was recorded after 3 h of induction.
B. Plate assay. Strain P. putida Pu→GFP (Table 1) bearing plasmid pCON922 (xylR3⁺) was spread on an agar plate sprinkled with solid TNT. Note fluorescence only in the close proximity of the compound. No other XylR variant or the wild‐type regulator was observed to produce a significant signal in the same conditions.