Aerobic Growth of Escherichia coli with 2,4,6-Trinitrotoluene (TNT) as the Sole Nitrogen Source and Evidence of TNT Denitration by Whole Cells and Cell-Free Extracts

Abstract

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...

Effect of 2,4,6-trinitrotoluene on soil bacterial communities

“…However, these amino derivatives tend to accumulate in the environment and are not degraded further. More interesting, a few bacteria have been reported to denitrate TNT [10,40] with the production of metabolites known to be easily mineralized [34]. Among those, a new Pseudomonas aeruginosa strain was recently isolated by Eyers et al from the soil KX1 (manuscript submitted).…”

“…OK-5 (Xanthomonadaceae), which is able to resist TNT-mediated stress by producing stress shock proteins [19] and to grow on TNT as sole nitrogen source [35], no other member of the families Comamonadaceae, Xanthomonadaceae and Caulobacteraceae has been reported so far to metabolize TNT. Indeed, bacteria known to degrade TNT aerobically and/or anaerobically belong to Pseudomonadaceae, Enterobacteriaceae, Mycobacteriaceae, Nocardiaceae, Desulfovibrionaceae, Clostridiaceae and Bacillaceae [36,40,41]. The presence of species belonging to Comamonadaceae, Xanthomonadaceae and Caulobacteraceae in soils with very high concentrations of TNT, where pore water is very probably saturated with TNT [44], suggests that those new species are at least tolerant to the presence of TNT.…”

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

“…In the course of our attempts to clone and identify other genes of Pseudomonas JLR11 encoding proteins involved in TNT metabolism, we designed a cloning strategy using Escherichia coli as a host strain. The series of controls indicated that E. coli was able to use TNT as the sole nitrogen source, thereby confirming recent findings (Stenuit et al ., 2006). In fact, it has long been known that E. coli cells growing aerobically or anaerobically are able to reduce up to two of the nitro groups of TNT to yield 2,4‐diamino‐6‐nitrotoluene (McCormick et al ., 1976; Ederer et al ., 1997; Fuller and Manning, 1997).…”

Molecular Tools for Monitoring and Validating Bioremediation

“…Occasionally, even negative results of screening a metagenomic library because of an ill-adapted host organism can be valuable, as illustrated by our recent discovery of strong TNT denitration capacity in Escherichia coli, the host par excellence, while screening for TNT denitration activity a metagenomic library which we established using DNA from soil historically contaminated with this recalcitrant explosive (Stenuit et al 2006). In this same context, valuable insights about shifts in microbial community structure as a result of TNT contamination were recently obtained by us using microarray probing as pointed out above and fingerprinting techniques (George et al 2008).…”