Engineering Pseudomonas fluorescens for Biodegradation of 2,4-Dinitrotoluene

MARIELA R. MONTI; ANDREA M. SMANIA; GEORGINA FABRO; MARIA E. ALVAREZ; CARLOS E. ARGARANA

APPLIED AND ENVIRONMENTAL MICROBIOLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington, USA; Año: 2005 vol. 71 p. 8864 - 8872

0099-2240

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerantrhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation ofthis priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer fromBurkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluenedegradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, andenergy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluenedegradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into theP. fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmidsystem, generated a degrading strain that was stable for a long time, which was designated RE. This strain wasable to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as acosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading2,4-dinitrotoluene at temperatures as low as 10°C. Finally, the presence of P. fluorescens RE in soils containinglevels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound onArabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic forA. thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibitedA. thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmentalrobustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.