Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.     

Engineering plants for the phytodetoxification of explosives

Summary  Widespread contaimination of the environment by explosives resulting from the manufacture, disposal and testing of munitions is becoming a matter of increasing concern. Most explosives are considered to be a major hazard to biological systems due to their toxic and mutagenic effects. Interest on the bioremediation of land contaminated with explosives has recently been focused on phytoremediation. Unfortunately., whilst plants have many advantages for the remediation of contaminated land and water, they lack the catabolic versatility which enables microorganisms to mineralize such a wide diversity of xenobiotic compounds. This raised the interesting question as to whether the impressive biodegradative capabilities of soil bacteria could be combined with the high biomass and stability of plants to yield an optimal system for in situ bioremediation of explosive residues in soil. Our investigation into the degradation of explosive residues by soil bacteria resulted in the isolation of Enterobacter cloacae PB2, which is capable of utilizing nitrate ester explosives such as pentaerythritol tetranitrate (PETN) and nitroglycerin as the sole source of nitrogen for growth. We have successfully introduced PETN reductase, the enzyme initiating explosive degradation in this organism, into plants to create transgenic plants that degrade explosives. Since the bacterial degradative pathways for many classes of organic pollutant have been elucidated, this may be a generally applicable method of achieving bioremediation of contaminated soil in the environment.     

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)

Pseudomonas putida JLR11 can grow on 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. We created nasB (nitrite reductase), pnrA (nitroaromatic reductase) and pnrA nasB mutants and tested their growth with TNT as the sole N source. The nasB and pnrA mutants grew at a reduced rate on TNT, whereas the double nasB pnrA mutant did not. This suggests that P. putida JLR11 carries out multiple enzymatic attacks on TNT-releasing nitrite and/or ammonium. The PnrA nitroreductase plays a key role in the reduction of TNT to 2,6-dinitro-4-hydroxylaminotoluene and the subsequent release of ammonium for growth.     

Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation

2,4,6-Trinitrotoluene (¹⁵N or ¹³C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [¹⁵N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [¹³C]TNT cultures and was related to Lysobacter taiwanensis.It has been estimated that there are over 1 million cubic yards of material contaminated with 2,4,6-trinitrotoluene (TNT) in the United States at concentrations as high as 600,000 to 700,00 mg/kg of material (9). Marine and estuarine sediments have also been impacted through the manufacturing, use, and/or disposal of TNT. Microbial biodegradation of these pollutants in situ is preferable due to the large volume of contaminated soils/sediments. However, it is unclear whether in situ bacteria can utilize TNT as a nitrogen or carbon source. Under aerobic conditions, TNT appears to be largely unavailable to bacteria but can be used by a variety of fungi as a carbon and nitrogen source (7). Under anaerobic conditions, only a few bacterial strains (Clostridium and Desulfovibrio strains and Pseudomonas sp. strain JLR11) have been reported to utilize TNT as a sole nitrogen source (6, 7). It is widely believed that nitroaromatic compounds cannot serve as growth substrates under anaerobic conditions in situ (11), and coamendment strategies are suggested for stimulating TNT transformation to 2,4,6-triaminotoluene (TAT) (1, 7, 18). Given these difficulties, there is no direct evidence that TNT can be biodegraded in situ and there is little proof that anaerobic bacteria can utilize TNT as a sole carbon or nitrogen source in organic-rich sediments. This study tested whether bacteria in Norfolk Harbor sediment are able to incorporate nitrogen (N) or carbon (C) from TNT into biomass under sulfidogenic conditions using stable-isotope probing (SIP). The findings indicate that bacteria assimilate ¹⁵N and ¹³C from TNT into their genomes during anaerobic incubations (2 to 35 days). Interestingly, one small-subunit (SSU) gene, related to Lysobacter taiwanensis, was observed in both the ¹⁵N and the ¹³C incubations.     

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

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-¹⁵N-labeled TNT.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

High TNT-transforming activity by a mixed culture acclimated and maintained on crude-oil-containing media

A mixed microbial culture originating from a petroleum-contaminated site and maintained on crude oil exhibited high 2,4,6-trinitrotoluene (TNT) transformation activity. Cultivation of the mixed culture in glucose-containing medium for 29 h resulted in almost complete transformation of 100 ppm TNT. TNT transformation was observed with both growing and resting cells. With subculturing, it was found that TNT could support growth of the mixed culture when supplied as sole carbon source, sole nitrogen source, or sole carbon and nitrogen source. The finding that a mixed microbial culture maintained on crude oil exhibited high TNT transformation activity without prior subculture on TNT-containing media is novel and may have potential practical applications in the bioremediation of munitions-contaminated soil and wastewater.     

Characterization of the plant growth promoting bacterium,Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa

The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future.     

Application of Bacillus sp. strain VT-8 for decontamination of TNT-polluted sites

Bacterial strain Bacillus sp. VT-8 was shown to use trinitrotoluene (TNT) as the sole source of carbon, nitrogen, and energy, as well as to carry out its co-oxidation. Resistance of Bacillus sp. VT-8 to high TNT concentrations was shown. Efficient detoxification of TNT-contaminated soil and water samples was demonstrated. This strain may be recommended for TNT biodegradation at concentrations of up to 140 mg/L due to its high degradation activity and the absence of toxic effect of TNT on Bacillus sp. VT-8.     

Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

The ability of Pseudomonas syringae pv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant, P. syringae ΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasB mutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting that P. syringae experiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-based P. syringae nitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation of P. syringae consumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy     

Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds

Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation.     

Initial Reductive Reactions in Aerobic Microbial Metabolism of 2,4,6-Trinitrotoluene

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H⁻-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H⁻-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT⁻), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.     

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp. (B strain) isolated from an anaerobic digester

A sulfate-reducing bacterium (SRB),Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327–332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO₂. This study demonstrated the possibility of using this isolated to decontaminate the soil and water contaiminated with TNT under anaerobic conditions.     

Biodegradation of 2-Chlorobenzoic Acid by Enterobacter cloacae: Growth Kinetics and Effect of Growth Conditions

Enterobacter cloacae was originally isolated from soil irrigated with wastewater on the basis of its ability to grow with linear alkylbenzene sulfonate (LAS) as the sole source for carbon and energy. The isolated bacterium was grown in batch cultures using a 2-chlorobenzoic acid (2-CBA)-containing minimal salt medium (MSM). 2-CBA was found to be the sole source for carbon and energy. 2-CBA inhibited the growth rate with a maximum concentration of 10 mM, after which no growth occurred. The Haldane model was used to predict the specific growth rate concentration data. 2-CBA degradation by starved E. cloaca cells was faster than that of nonstarved cells. The maximum growth rates on 2-CBA (2 mM) for starved and nonstarved cells reached only 0.34 and 0.28 h^?1, respectively. Glucose, lactose, sucrose, maltose, succinic acid, and mannitol as additional carbon sources at a fixed concentration (0.2%) caused the degradation rate of 2-CBA to proceed faster at ranges between 1.08- and 1.5-fold higher than that of the control. In contrast, using only fructose and sorbitol as the carbon sources showed catabolic repression of the degradation activity of 2-CBA by E. cloaca cells, although their cell mass was improved. All nitrogen sources supplied caused an increase in cell mass, whereas only lysine, alanine, glutamine, casein, and yeast extract caused a decrease in the degradation rate of 2-CBA, with a range between 12% and 28%. The activity of C120 could be detected in a crude extract of E. cloacae cells, indicating that the chloroaromatic ring fission occurs through the ortho pathways, not through the meta pathways. The data showed that different initial cell (inocula) densities did not affect the induction time for 2-CBA degradation. However, doubling the initial cell densities reduced the time required for reaching the complete degradation. 2-CBA degradation was optimally achieved at a 37°C incubation temperature and a pH of 7.5.     

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Δ¹-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.