Engineering Pseudomonas fluorescens for biodegradation of the xenobiotic 2,4-dinitrotoluene

MONTI MARIELA R.; FABRO GEORGINA; ALVAREZ MARÍA E.; SMANIA ANDREA. M.; ARGARAÑA CARLOS E.

Córdoba-Argentina

Workshop; Argentine Workshop on current topics in Pseudomonas and Burkholderia research; 2007

In the present work, the non-pathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of the priority pollutant 2,4-dinitrotoluene (2,4-DNT).  Firstly, a recombinant strain named MP, was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid which contains the dnt genes for 2,4-DNT degradation. This strain was able to grow on 2,4-DNT as the sole source of carbon, nitrogen, and energy at equivalent levels as Burkholderia sp. strain DNT. Nevertheless, the acquired 2,4-DNT degradative phenotype of pJS1-borne P. fluorescens MP was lost in the absence of 2,4-DNT. The introduction of dnt genes into the P. fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a long stable degrading strain, named RE. This strain was able to use 2,4-DNT as a sole nitrogen source and to completely degrade this compound as cosubstrate. Furthermore, P. fluorescens RE was capable of degrading 2,4-DNT at temperatures as low as 10ºC whereas Burkholderia DNT was unable to sustain a complete degradation process accumulating toxic 2,4-DNT intermediates. The presence of P. fluorescens RE in soils containing plant lethal levels of 2,4-DNT significantly decreased the toxic effects of this nitrocompound on Arabidopsis thaliana growth. Finally, P. fluorescens RE was found to be harmlessness for the plant species A. thaliana and Nicotiana tabacum, whereas Burkholderia sp. strain DNT inhibited A. thaliana seed germination and produced plant lethality. These results provide evidences that the use of a harmless and psychrotolerant rhizobacteria host for the transference of 2,4-DNT degradative ability is an interesting approach for the generation of biological vehicles for cleaning environments contaminated with this priority pollutant.