Jim C. Spain

Biodegradation of nitroaromatic compounds

Nitroaromatic compounds are released into the biosphere almost exclusively from anthropogenic sources. Some compounds are produced by incomplete combustion of fossil fuels; others are used as synthetic intermediates, dyes, pesticides, and explosives. Recent research revealed a number of microbial systems capable of transforming or biodegrading nitroaromatic compounds. Anaerobic bacteria can reduce the nitro group via nitroso and hydroxylamino intermediates to the corresponding amines. Isolates of Desulfovibrio spp. can use nitroaromatic compounds as their source of nitrogen. They can also reduce 2,4,6-trinitrotoluene to 2,4,6-triaminotoluene. Several strains of Clostridium can catalyze a similar reduction and also seem to be able to degrade the molecule to small aliphatic acids. Anaerobic systems have been demonstrated to destroy munitions and pesticides in soil. Fungi can extensively degrade or mineralize a variety of nitroaromatic compounds. For example, Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene and 2,4,6-trinitrotoluene and shows promise as the basis for bioremediation strategies. The anaerobic bacteria and the fungi mentioned above mostly transform nitroaromatic compounds via fortuitous reactions. In contrast, a number of nitroaromatic compounds can serve as growth substrates for aerobic bacteria. Removal or productive metabolism of nitro groups can be accomplished by four different strategies. (a) Some bacteria can reduce the aromatic ring of dinitro and trinitro compounds by the addition of a hydride ion to form a hydride-Meisenheimer complex, which subsequently rearomatizes with the elimination of nitrite. (b) Monooxygenase enzymes can add a single oxygen atom and eliminate the nitro group from nitrophenols. (c) Dioxygenase enzymes can insert two hydroxyl groups into the aromatic ring and precipitate the spontaneous elimination of the nitro group from a variety of nitroaromatic compounds. (d) Reduction of the nitro group to the corresponding hydroxylamine is the initial reaction in the productive metabolism of nitrobenzene, 4-nitrotoluene, and 4-nitrobenzoate. The hydroxylamines undergo enzyme-catalyzed rearrangements to hydroxylated compounds that are substrates for ring-fission reactions. Potential applications of the above reactions include not only the biodegradation of environmental contaminants, but also biocatalysis and synthesis of valuable organic molecules.

Enzyme immobilization in a biomimetic silica support

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure. Here we show that butyrylcholinesterase entrapped during the precipitation of silica nanospheres retained all of its activity. Ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme. The mechanical properties of silica nanospheres facilitated application in a flow-through reactor. The use of biosilica for enzyme immobilization combines the excellent support properties of a silica matrix with a benign immobilization method that retains enzyme activity.

Biodegradation of nitroaromatic compounds and explosives

Introduction, J.C. Spain Strategies for Aerobic Degradation of Nitroaromatic Compounds by Bacteria: Process Discovery to Field Application, S.F. Nishimo, J.C. Spain, and Z. He Molecular Biology of Nitroarene Degradation, R.E. Parales Perspectives of Bioelimination of Polynitroaromatic Compounds, H. Lenke, C. Achtnich, and H-J. Knackmuss Identification of Genes Involved in Picric Acid and 2,4-Dinitrophenol Degradation by mRNA Differential Display, R. Ross, D.M. Walters, H-J. Knackmuss, and P.E. Rouviere Microbial Degradation of Mononitrophenols and Mononitrobenzoates, G.J. Zylstra, S-W. Bang, L.M. Newman, and L.L. Perry The Role of Nitrate Ester Reductase Enzymes in the Biodegradation of Explosives, R.E. Williams and N.C. Bruce Anaerobic Transformation of TNT by Clostridium, F. Ahmad and J.B. Hughes Fungal Degradation of Explosives: TNT and Related Nitroaromatic Compounds, W. Fritsche, K. Scheibner, A. Herre, and M. Hofrichter Phytoremediation and Plant Metabolism of Explosives and Nitroaromatic Compounds, J.G. Burken, J.V. Shanks, and P.L. Thompson Biodegradation of RDX and HMX: From Basic Research to Field Application, J. Hawari Subsurface Chemistry of Nitroaromatic Compounds, S.B. Haderlein, T.B. Hofstetter, and R.P. Schwartzenbach Composting (Humification) of Nitroaromatic Compounds, D. Bruns-Nagel, K. Steinbach, D.Gemsa, and E. von Low Application and Costs for Biological Treatment of Explosive-Contaminated Soils in the United States, D.E. Jerger and P.M. Woodhull

Biodegradation of cis-Dichloroethene as the Sole Carbon Source by a β-Proteobacterium

ABSTRACT An aerobic bacterium capable of growth on cis-dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata, indicating that the isolate was a β-proteobacterium. At 20°C, strain JS666 grew on cDCE with a minimum doubling time of 73 ± 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 ± 0.2 μM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride, trans-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.

Phylogenetic and Kinetic Diversity of Aerobic Vinyl Chloride-Assimilating Bacteria from Contaminated Sites

ABSTRACT Aerobic bacteria that grow on vinyl chloride (VC) have been isolated previously, but their diversity and distribution are largely unknown. It is also unclear whether such bacteria contribute to the natural attenuation of VC at chlorinated-ethene-contaminated sites. We detected aerobic VC biodegradation in 23 of 37 microcosms and enrichments inoculated with samples from various sites. Twelve different bacteria (11 Mycobacterium strains and 1 Nocardioides strain) capable of growth on VC as the sole carbon source were isolated, and 5 representative strains were examined further. All the isolates grew on ethene in addition to VC and contained VC-inducible ethene monooxygenase activity. The Mycobacterium strains (JS60, JS61, JS616, and JS617) all had similar growth yields (5.4 to 6.6 g of protein/mol), maximum specific growth rates (0.17 to 0.23 day−1), and maximum specific substrate utilization rates (9 to 16 nmol/min/mg of protein) with VC. The Nocardioides strain (JS614) had a higher growth yield (10.3 g of protein/mol), growth rate (0.71 day−1), and substrate utilization rate (43 nmol/min/mg of protein) with VC but was much more sensitive to VC starvation. Half-velocity constant (Ks) values for VC were between 0.5 and 3.2 μM, while Ks values for oxygen ranged from 0.03 to 0.3 mg/liter. Our results indicate that aerobic VC-degrading microorganisms (predominantly Mycobacterium strains) are widely distributed at sites contaminated with chlorinated solvents and are likely to be responsible for the natural attenuation of VC.

Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

ABSTRACT Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO2−) (30%), nitrous oxide (N2O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [15N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N2O with two molecular mass ions: one at 44 Da, corresponding to 14N14NO, and the second at 45 Da, corresponding to 15N14NO. The nonlabeled N2O could be formed only from -NO2, whereas the 15N-labeled one was presumed to originate from a nitramine group (15N-14NO2) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C2H5N3O3. When the experiment was repeated with ring-labeled [15N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-14C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in 14CO2 (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO2 and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.

Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

ABSTRACT An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway.Burkholderia cepacia strain JS850 andHydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.

The COMBREX Project: Design, Methodology, and Initial Results

Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.

Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from Comamonas sp. Strain JS765

ABSTRACT Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated ReductaseNBZ, FerredoxinNBZ, OxygenaseNBZα, and OxygenaseNBZβ, with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified OxygenaseNBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.

Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

ABSTRACT A unique metabolite with a molecular mass of 119 Da (C2H5N3O3) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.