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

Abstract

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.

Figure 1

Growth yield of P. putida KT2440 and several isogenic mutants in TNT‐free and TNT‐saturated medium. Cultures were inoculated at ∼0.05 OD₆₆₀ and incubated at 30°C for 24 h with shaking (150 r.p.m.) in a Kühner incubator. Grey bars represent growth in TNT‐free M9 minimal medium. Black bars represent growth in TNT‐saturated glucose‐supplemented M9 minimal medium.