A double mutant of <i>Pseudomonas putida</i> JLR11 deficient in the synthesis of the nitroreductase PnrA and assimilatory nitrite reductase NasB is impaired for growth on 2,4,6‐trinitrotoluene (TNT)

Caballero, Antonio; Ramos, Juan L.

doi:10.1111/j.1462-2920.2006.01012.x

Cited by 23 publications

(16 citation statements)

References 32 publications

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“…The nitroreductases NfsA and NfsB, together with the N-ethylmaleimide reductase NemA, contributed to the ability of E. coli AB1157 to obtain usable nitrogen from TNT (51). Similarly, the nitroreductase PrnA was shown to be involved in the utilization of TNT as a nitrogen source by P. putida JLR11, and the assimilatory nitrite reductase NasB contributed to the ability of the strain to grow efficiently (14,15). A mechanism for the release of nitrite during the condensation of hydroxylaminodinitrotoluene (the product of nitroreductase activity on TNT) and a Meisenheimer dihydride complex (Fig.…”

Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

“…Two Gram-positive isolates, strains TNT-8 and TNT-32, were able to use TNT as a nitrogen source, but the mechanism of nitrogen assimilation remains unclear (200). More recently, Ramos et al reported that Pseudomonas putida JLR11 (13)(14)(15) and Escherichia coli AB1157 (51) were able to use TNT as a nitrogen source for growth, reducing the nitro group and recovering the ammonium by the use of nitroreductases. The nitroreductases NfsA and NfsB, together with the N-ethylmaleimide reductase NemA, contributed to the ability of E. coli AB1157 to obtain usable nitrogen from TNT (51).…”

Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

“…A mechanism for the release of nitrite during the condensation of hydroxylaminodinitrotoluene (the product of nitroreductase activity on TNT) and a Meisenheimer dihydride complex (Fig. 12B) of TNT to form a diarylamine was proposed based on studies with 15 N-labeled TNT (207).…”

Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

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Nitroaromatic Compounds, from Synthesis to Biodegradation

Parales

2010

Microbiol Mol Biol Rev

744

470

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SUMMARY Nitroaromatic compounds are relatively rare in nature and have been introduced into the environment mainly by human activities. This important class of industrial chemicals is widely used in the synthesis of many diverse products, including dyes, polymers, pesticides, and explosives. Unfortunately, their extensive use has led to environmental contamination of soil and groundwater. The nitro group, which provides chemical and functional diversity in these molecules, also contributes to the recalcitrance of these compounds to biodegradation. The electron-withdrawing nature of the nitro group, in concert with the stability of the benzene ring, makes nitroaromatic compounds resistant to oxidative degradation. Recalcitrance is further compounded by their acute toxicity, mutagenicity, and easy reduction into carcinogenic aromatic amines. Nitroaromatic compounds are hazardous to human health and are registered on the U.S. Environmental Protection Agency's list of priority pollutants for environmental remediation. Although the majority of these compounds are synthetic in nature, microorganisms in contaminated environments have rapidly adapted to their presence by evolving new biodegradation pathways that take advantage of them as sources of carbon, nitrogen, and energy. This review provides an overview of the synthesis of both man-made and biogenic nitroaromatic compounds, the bacteria that have been identified to grow on and completely mineralize nitroaromatic compounds, and the pathways that are present in these strains. The possible evolutionary origins of the newly evolved pathways are also discussed.

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Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

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Nitroaromatic Compounds, from Synthesis to Biodegradation

Parales

2010

Microbiol Mol Biol Rev

744

470

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“…These compounds are susceptible to the reduction of the nitro side groups to yield hydroxylamino groups. This type of activity is also called nitroreductase activity (6,7,8,9,20,28,34) and will be referred to here as type I hydride transferase activity. Other OYE members have been described to additionally catalyze the nucleophilic attack on the aromatic ring of 2,4,6-trinitrotoluene (TNT) by hydride ions to produce Meisenheimer monohydride and dihydride complexes (13,23,24,29,32,37).…”

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confidence: 99%

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

Dillewijn

Wittich

Caballero

et al. 2008

Appl Environ Microbiol

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To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.

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“…Since E. coli is unable to assimilate nitrate or nitrite under aerobic conditions (12), the use of nitrogen from TNT occurred via a metabolic pathway other than the direct release of nitrite. Recent reports have postulated the partial reduction of one or more nitro groups of TNT to its hydroxylamino moiety and the subsequent release of ammonium from the aromatic ring (3)(4)(5)16), probably through a Bamberger-like rearrangement (11). Next, ammonium was likely used as a nitrogen source by E. coli via the glutamine synthetase/glutamate synthase pathway (16).…”

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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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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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A double mutant of Pseudomonas putida JLR11 deficient in the synthesis of the nitroreductase PnrA and assimilatory nitrite reductase NasB is impaired for growth on 2,4,6‐trinitrotoluene (TNT)

Cited by 23 publications

References 32 publications

Nitroaromatic Compounds, from Synthesis to Biodegradation

Nitroaromatic Compounds, from Synthesis to Biodegradation

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

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

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