Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.

Escherichia coli grew aerobically with 2,4,6-trinitrotoluene (TNT) as sole nitrogen source and caused TNT's partial denitration. This reaction was enhanced in nongrowing cell suspensions with 0.516 mol nitrite released per mol TNT. Cell extracts denitrated TNT in the presence of NAD(P)H. Isomers of amino-dimethyl-tetranitrobiphenyl were detected and confirmed with U-15 N-labeled TNT.2,4,6-Trinitrotoluene (TNT) is recalcitrant to microbial degradation. Denitration (defined as the release of nitrite) is a critical step for further mineralization of TNT (19). A welldescribed TNT denitration pathway involves a nucleophilic addition of hydride ions to the aromatic ring with subsequent nitrite release. Three enzymes performing this addition in the presence of NAD(P)H have been characterized so far: pentaerythritol tetranitrate reductase of Enterobacter cloacae PB2 (9), xenobiotic reductase B (XenB) of Pseudomonas fluorescens I-C (15), and N-ethylmaleimide (NEM) reductase of Escherichia coli (21). In vitro denitration of TNT with purified NEM reductase was described by Williams et al. (21), but the authors did not provide quantitative data with E. coli cells. Also, several reports have described the reduction of TNT by E. coli but not its denitration (6,14,22,23). Only one recent study has mentioned denitration of TNT by E. coli, but no quantitative data were provided and TNT was not the sole nitrogen source (13). The objectives of this study were to determine the kinetics of TNT denitration by E. coli and identify TNT denitrated metabolites.E. coli strains EPI300 (Epicentre Technologies, Madison, WI) and LK111 (24) were routinely cultivated at 37°C in LuriaBertani (LB) broth. Cells were harvested at mid-exponential phase and washed three times with phosphate-buffered saline (containing, per liter, 7 g of Na 2 HPO 4 ·12H 2 O, 3 g of KH 2 PO 4 , 1 g of NaCl). Cells were resuspended in 20 ml of phosphatebuffered saline and used for TNT biodegradation assays.TNT was obtained from Nobel Explosives (Châtelet, Belgium) and was 99.5% pure by high-performance liquid chromatography. Growing cell experiments were carried out in modified mineral salts medium (10) containing 20 mM of glycerol or glucose and TNT as the sole nitrogen source. The medium was inoculated at an optical density at 600 nm (OD 600 ) of 0.025 and incubated at 37°C and 250 rpm. Controls consisted of flasks without TNT, flasks without cells, flasks without a carbon source, and flasks with 200 ppm of Hg 2 Cl 2 and without a carbon source. Nitrite, TNT, and metabolites were quantitatively determined as previously described (7).Bacterial growth of E. coli EPI300 was observed with glycerol and TNT (606 M on the basis of high-performance liquid chromatography analysis) as the sole nitrogen source (Fig. 1A). The growth was relatively fast over the first 26 h, reaching a plateau of 0.120 OD 600 units after 117 h of incubation. With glucose and TNT (588 M), the bacterial growth profile was similar but the OD 600 reached 0.320 after 117 h (Fig. 1B). Without TNT, no si...

To gain insight into the impact of 2,4,6-trinitrotoluene (TNT) on soil microbial communities, we characterized the bacterial community of several TNT-contaminated soils from two sites with different histories of contamination and concentrations of TNT. The amount of extracted DNA, the total cell counts and the number of CFU were lower in the TNT-contaminated soils. Analysis of soil bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soils, as well as the presence of Caulobacteraceae. CFU from TNT-contaminated soils were identified as Pseudomonadaceae, and, to a lesser extent, Caulobacteraceae. Finally, a pristine soil was spiked with different concentrations of TNT and the soil microcosms were incubated for 4 months. The amount of extracted DNA decreased in the microcosms with a high TNT concentration [1.4 and 28.5 g TNT/kg (dry wt) of soil] over the incubation period. After 7 days of incubation of these soil microcosms, there was already a clear shift of their original flora towards a community dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. These results indicate that TNT affects soil bacterial diversity by selecting a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae.

2,4,6-trinitrotoluene (TNT) is known to be one of the most common military explosives. In spite of its established toxicity and mutagenicity for many organisms, soils and groundwater are still being frequently contaminated at manufacturing, disposal and TNT destruction sites. The inability of natural aquatic and soil biota to use TNT as growth substrate has been recognized as the primary limitation in the application of bioremediation processes to contaminated environments. However, promising degradation pathways have been recently discovered which may lead to the mineralisation of TNT. Significant advances have been made in studying the mechanism of TNT denitration, which can be considered as the major reaction and the driving force towards beneficial biodegradation. The possibilities to favour TNT denitration are discussed based on current knowledge of the enzymology and genetics of denitration in nitroaromatic degrading organisms. The literature survey demonstrates that the only enzymes characterized so far for their denitrase activity towards TNT belong to the class I flavin-dependent b/a barrel oxidoreductases, also known as the ''Old Yellow Enzyme'' family. In addition, this review provides an overview of strategies and future directions towards a rational search for new catabolic activities, including metagenomic library screening, plus new possibilities to improve the activity of known catabolic enzymes acting on TNT, such as DNA shuffling.

The denitration of 2,4,6-trinitrotoluene (TNT) can produce mono- or dinitro aromatic compounds susceptible to microbial mineralization. In the present study, denitration of TNT and other nitro aromatic compounds was investigated with a solid-phase extract obtained from the culture supernatant of Pseudomonas aeruginosa ESA-5 grown on a chemically defined aerobic medium. When the C18 solid-phase extract containing extracellular catalysts (EC) was incubated with TNT and NAD(P)H, we observed a significant release of nitrite. The concentration of nitrite released in the reaction medium was strongly dependent on the concentration of NAD(P)H and EC. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde, and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites, and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitro aromatic parent molecule. During this process, the production of toxic reduced TNT metabolites was minimal. The incubation of EC with TNT, NAD(P)H, and specific scavengers of reactive oxygen species suggested the involvement of superoxide radicals (O2*-) and hydrogen peroxide in the denitration process. Results obtained in this study reveal for the first time that extracellular small-molecular-weight substance(s) of bacterial origin can serve as green catalyst(s) to initiate TNT denitration. In addition, this study gives clear evidence for the production of a TNT metabolite bearing a single nitro groupfollowing a denitration reaction with catalyst(s) of biotic origin.