Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Yin, Hong; Wood, Thomas K.; Smets, Barth F.

doi:10.1007/s00253-005-1988-0

Cited by 10 publications

(12 citation statements)

References 27 publications

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“…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).…”

mentioning

confidence: 99%

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

Stenuit¹,

Eyers²,

Rozenberg

et al. 2006

Appl Environ Microbiol

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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...

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confidence: 99%

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

Stenuit¹,

Eyers²,

Rozenberg

et al. 2006

Appl Environ Microbiol

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“…Several bacterial species with the ability to reduce TNT nitro groups have been described [ 1 , 3 , 26 ]. E. coli has proven ability to transform TNT to ADNTs and DANTs under both aerobic and anaerobic growth conditions [ 13 , 30 , 32 , 47 , 48 ]. TAT has also been detected in cell extract of E. coli grown anaerobically under a hydrogen atmosphere [ 49 ].…”

Section: Resultsmentioning

confidence: 99%

“…Particularly, a large body of research is devoted to isolation and characterization of TNT-degrading microbes [ 3 , 6 , 9 , 13 , 26 ]. E. coli is one of the microorganisms that has shown remarkable TNT biotransformation capability [ 27 , 28 , 29 , 30 ]. E. coli possesses multiple enzymes to attack TNT, including nitroreductases capable of reducing the TNT nitro groups and N-ethylmaleimide (NEM) involved in TNT denitration [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, in the present study we used CBA to profile the metabolic capacity of E. coli in biotransformation of TNT. In doing so, we extended the most comprehensive high quality GSMM of E. coli , iJO1336 [ 43 ], to account for the known or evidently hypothesized pathways involving in TNT degradation by this organism [ 1 , 2 , 3 , 9 , 13 , 27 , 28 , 29 , 30 , 44 , 45 ]. The extended model was then analysed by a set of CBA methods [ 37 ], including flux balance analysis (FBA), flux variability analysis (FVA), Monte Carlo flux sampling [ 46 ], robustness analysis (RA), and phenotype phase plane (PhPP) analysis for network-level characterization of TNT transformation in E. coli .…”

Section: Introductionmentioning

confidence: 99%

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Systems Biology Approach to Bioremediation of Nitroaromatics: Constraint-Based Analysis of 2,4,6-Trinitrotoluene Biotransformation by Escherichia coli

et al. 2017

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Microbial remediation of nitroaromatic compounds (NACs) is a promising environmentally friendly and cost-effective approach to the removal of these life-threating agents. Escherichia coli (E. coli) has shown remarkable capability for the biotransformation of 2,4,6-trinitro-toluene (TNT). Efforts to develop E. coli as an efficient TNT degrading biocatalyst will benefit from holistic flux-level description of interactions between multiple TNT transforming pathways operating in the strain. To gain such an insight, we extended the genome-scale constraint-based model of E. coli to account for a curated version of major TNT transformation pathways known or evidently hypothesized to be active in E. coli in present of TNT. Using constraint-based analysis (CBA) methods, we then performed several series of in silico experiments to elucidate the contribution of these pathways individually or in combination to the E. coli TNT transformation capacity. Results of our analyses were validated by replicating several experimentally observed TNT degradation phenotypes in E. coli cultures. We further used the extended model to explore the influence of process parameters, including aeration regime, TNT concentration, cell density, and carbon source on TNT degradation efficiency. We also conducted an in silico metabolic engineering study to design a series of E. coli mutants capable of degrading TNT at higher yield compared with the wild-type strain. Our study, therefore, extends the application of CBA to bioremediation of nitroaromatics and demonstrates the usefulness of this approach to inform bioremediation research.

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“…Many microbes, including Escherichia coli [6][7][8], Enterobacter cloacae [9], Bacillus cereus [10], Clostridium acetobutylicum [11], Clostridium thermoaceticum [12], Klebsiella sp. C1 [13], Phanerochaete chrysosporium [14], Pseudomonas sp.…”

Section: Introductionmentioning

confidence: 99%

Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

Bai

Yang

Liu

et al. 2015

Process Biochemistry

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Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Cited by 10 publications

References 27 publications

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

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

Systems Biology Approach to Bioremediation of Nitroaromatics: Constraint-Based Analysis of 2,4,6-Trinitrotoluene Biotransformation by Escherichia coli

Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

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