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Applied and Environmental Microbiology, December 2005, p. 8864-8872, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8864-8872.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Engineering Pseudomonas fluorescens for Biodegradation of 2,4-Dinitrotoluene{dagger}

Mariela R. Monti, Andrea M. Smania,{ddagger} Georgina Fabro, María E. Alvarez, and Carlos E. Argaraña*{ddagger}

Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina

Received 4 June 2005/ Accepted 12 September 2005

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10°C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.

* Corresponding author. Mailing address: Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000, Córdoba, Argentina. Phone: 54-351-4334168. Fax: 54-351-4334074. E-mail: carga{at}mail.fcq.unc.edu.ar.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} A.M.S. and C.E.A. contributed equally to this article.

Applied and Environmental Microbiology, December 2005, p. 8864-8872, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8864-8872.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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