Bioremediation of 2,4,6-trinitrotoluene under field conditions.

In situ bioremediation of the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) provides a cost-effective alternative for cleaning up contaminated sites. Here we compare the effectiveness of several bioremediation techniques: natural attenuation, bioaugmentation with TNT-degrading Pseudomonas putida JLR11, phytoremediation with maize (Zea mays L.) and broad beans (Vicia faba L.), and rhizoremediation with maize and broad beans inoculated with P. putida JLR11. Experiments in spiked hydroponic medium demonstrated that inoculation with bacteria did not affect TNT levels. On the other hand, axenic plants were able to remove 32% to 38% of the TNT from the medium. However, when plants were inoculated with bacteria,TNT disappeared to an even greater extent (80% to 88%), a result that advocates a role for P. putida JLR11 in rhizoremediation. In field experiments neither natural attenuation nor bioaugmentation with P. putida JLR11 affected TNT levels to a significant degree. However, the extractable TNT content in rhizosphere soil associated to maize roots decreased by more than 96% in 60 days regardless of inoculation. This indicates that under these field conditions, the effect of phytoremediation by maize overshadowed any effect of rhizoremediation by P. putida JLR11.

Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth.

There has been a growing interest in the degradation of 2,4,6-trinitrotoluene (TNT) over the last decade, ever since its removal from polluted sites was declared an international environmental priority. Certain aerobic and anaerobic microorganisms are capable of using TNT as an N source, although very few studies have proven the mineralization of this compound. An unexpected observation in our laboratory led us to discover that certain Escherichia coli bench laboratory strains have multiple enzymes that attack TNT. One of the NemA products is responsible for the release of nitrite from the nitroaromatic ring: among the metabolites observed in vitro include Meisenheimer dihydride complexes of TNT from which 2-hydroxylamino-6-nitrotoluene is slowly formed during their rearomatization under concomitant release of nitrite. Furthermore, NemA, together with NfsA and NfsB reduce the nitro groups on the aromatic ring to the corresponding hydroxylamino derivatives, which probably results in the release of ammonium ions which can, in turn be used as a nitrogen source by E. coli for growth.

Bioremediation of polynitrated aromatic compounds: plants and microbes put up a fight.

Industrialization and the quest for a more comfortable lifestyle have led to increasing amounts of pollution in the environment. To address this problem, several biotechnological applications aimed at removing this pollution have been investigated. Among these pollutants are xenobiotic compounds such as polynitroaromatic compounds--recalcitrant chemicals that are degraded slowly. Whereas 2,4,6-trinitrophenol (TNP) can be mineralized and converted into carbon dioxide, nitrite and water, 2,4,6-trinitrotoluene (TNT) is more recalcitrant--although several microbes can use it as a nitrogen source. The most effective in situ biotreatments for TNT are the use of bioslurry (which can be preceded by an abiotic step) and phytoremediation. Phytoremediation can be enhanced by using transgenic plants alone or together with microbes.

Cellular XylS levels are a function of transcription of xylS from two independent promoters and the differential efficiency of translation of the two mRNAs.

XylS controls the expression of the meta-cleavage pathway for the metabolism of benzoates in Pseudomonas putida KT2440. The xylS gene is expressed from two promoters, Ps1 and Ps2. Transcription from Ps2 is low and constitutive, whereas transcription from Ps1 is induced in the presence of toluene. In this study, we also show that translation of mRNA generated from Ps1 is 10 times more efficient than that generated from Ps2. This pattern of transcription and translation of xylS gives rise to two modes of activation of the promoter of the meta pathway operon (Pm) according to the concentration of XylS in the cell. In cells growing with benzoate, with small amounts of XylS, the activated XylS regulator binds the effector and stimulates transcription from Pm, whereas in cells growing with toluene, the high levels of XylS suffice to stimulate transcription from Pm even in the absence of XylS effectors.

XylS activator and RNA polymerase binding sites at the Pm promoter overlap.

Transcription from the TOL plasmid meta-cleavage pathway operon, Pm, depends on the XylS protein being activated by a benzoate effector. The XylS binding sites are two imperfect 5'-TGCAN(6)GGNTA-3' direct repeats located between positions -70/-56 and -49/-35 [González-Pérez et al. (1999) J. Biol. Chem. 274, 2286-2290]. An intrinsic bending of 40 degrees, which is not essential for transcription, is centered at position -43. We have determined the potential overlap between the XylS and RNA polymerase binding sites. The insertion of 2 or more bp between C and T at positions -37 and -36 abolished transcription activation by the wild-type XylS and by XylSS229I, a mutant with increased affinity for the XylS binding sites. In contrast, a 1-bp insertion at -37 was permissible, although when in addition to the 1-bp insertion at -37 the mutant promoter had a point mutation at the XylS binding site (C-47-->T), transcription was abolished with the wild-type XylS protein, but not with XylSS229I. The overlap between the proximal XylS binding site and the -35 region recognized by RNA polymerase at positions -35 and -36 appears to be critical for transcription.