• Open Access

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6-trinitrotoluene

Authors

  • Matilde Fernández,

    1. Bio-Iliberis Research and Development, Edificio BIC, Parque Tecnológico de Ciencias de la Salud, E-18100 Armilla, Granada, Spain.
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  • Estrella Duque,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada. Spain.
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  • Paloma Pizarro-Tobías,

    1. Bio-Iliberis Research and Development, Edificio BIC, Parque Tecnológico de Ciencias de la Salud, E-18100 Armilla, Granada, Spain.
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  • Pieter Van Dillewijn,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada. Spain.
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  • Rolf-Michael Wittich,

    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada. Spain.
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  • Juan L. Ramos

    Corresponding author
    1. Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada. Spain.
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*E-mail jlramos@eez.csic.es; Tel. (+34) 958 181600; Fax (+34) 958 129600.

Summary

Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6-trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification-related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up- and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1-oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.

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