Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes.
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.
Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
Junkal
J
Garmendia
Aitor
A
de las Heras
Teca Calcagno
TC
Galvão
Víctor
V
de Lorenzo
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053834:Explosive Agents / Q000032:analysis* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000032:analysis* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014303:Trinitrotoluene / Q000032:analysis* / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000032:analysis / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D053834:Explosive Agents; D012989:Soil Pollutants; D014157:Transcription Factors; D014874:Water Pollutants, Chemical; C074322:XylR protein, Pseudomonas; D014303:Trinitrotoluene
J Mol Biol. 2007 Sep 21;372(3):571-82
17681542
Mol Microbiol. 2007 Aug;65(4):907-19
17645451
Appl Environ Microbiol. 2000 Jan;66(1):163-9
10618218
J Bacteriol. 1990 Nov;172(11):6557-67
2172216
Curr Opin Biotechnol. 2006 Feb;17(1):34-42
16359854
Mol Microbiol. 1996 Jan;19(1):7-17
8821932
Curr Opin Biotechnol. 2000 Oct;11(5):467-75
11024365
J Chromatogr A. 2002 Jul 19;963(1-2):411-8
12187997
Appl Microbiol Biotechnol. 2000 Jun;53(6):736-41
10919336
Annu Rev Microbiol. 1997;51:341-73
9343354
Environ Microbiol. 2002 Dec;4(12):799-808
12534463
Nat Chem Biol. 2007 Jun;3(6):325-30
17486045
J Environ Manage. 2004 Apr;70(4):291-307
15016438
Plasmid. 1997;38(1):35-51
9281494
Appl Environ Microbiol. 2005 Feb;71(2):883-92
15691944
J Mol Biol. 1990 Jan 20;211(2):373-82
2407853
Nature. 1994 Aug 4;370(6488):389-91
8047147
J Magn Reson. 1998 Dec;135(2):373-9
9878466
Mol Microbiol. 2001 Oct;42(1):47-59
11679066
Environ Microbiol. 2004 Oct;6(10):1005-20
15344926
J Bacteriol. 2007 Oct;189(19):6751-62
17660288
Appl Environ Microbiol. 1994 May;60(5):1487-94
8017932
J Bacteriol. 2000 Mar;182(5):1352-5
10671458
Appl Radiat Isot. 2004 Jul;61(1):11-20
15145433
Biosens Bioelectron. 2007 Aug 30;23(1):1-18
17662594
J Bacteriol. 1998 Feb;180(3):600-4
9457863
Nature. 2003 May 8;423(6936):185-90
12736688
Environ Microbiol. 2003 Dec;5(12):1309-27
14641576
J Bacteriol. 1994 Dec;176(24):7550-7
8002579
J Bacteriol. 1994 Aug;176(16):5052-8
8051017
Biodegradation. 2000;11(1):37-47
11194972
Mol Microbiol. 1995 Apr;16(2):205-13
7565083
Anal Chem. 2000 Dec 1;72(23):5834-40
11128944
J Mol Biol. 1996 May 17;258(4):562-74
8636992
MAGMA. 1999 Dec;9(3):103-8
10628681
Nature. 2005 Nov 24;438(7067):449-53
16306983
Chem Rev. 2000 Jul 12;100(7):2705-38
11749302
Curr Opin Microbiol. 2003 Jun;6(3):310-6
12831909
Biosens Bioelectron. 2004 May 15;19(10):1319-23
15046765
J Mol Biol. 1996 May 17;258(4):575-87
8636993
J Bacteriol. 1994 Mar;176(6):1555-60
8132448
Appl Radiat Isot. 2000 Oct-Nov;53(4-5):557-63
11003491
Methods Enzymol. 1994;235:386-405
8057911
J Mol Biol. 2004 Jul 9;340(3):405-18
15210343
Appl Environ Microbiol. 2004 Jan;70(1):508-17
14711682
Mol Gen Genet. 1992 May;233(1-2):293-301
1318499
Gene. 1997 Jun 3;191(2):149-53
9218713
J Bacteriol. 2000 Jun;182(11):3008-16
10809676
Microbiol Mol Biol Rev. 2004 Sep;68(3):474-500, table of contents
15353566