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 and Hydrogenophaga 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.2,6-Dinitrotoluene (2,6-DNT) and 2,4-dinitrotoluene (2,4-DNT) occur as soil and groundwater contaminants at former 2,4,6-trinitrotoluene (TNT) production sites and in the wastewater from the commercial production of feedstocks for polyurethane foam (23). Twenty years after the cessation of TNT production in the United States, the manufacturing sites are still heavily contaminated with both 2,4-and 2,6-DNT even though 2,4-DNT-mineralizing bacteria can be readily isolated from the contaminated material (26). Commercial manufacture of DNT results in the release of DNT to industrial and municipal waste treatment systems (information found at the Environmental Health Center website [http://safety.webfirst .com/ehc/ew/chemical.htm] and in the TOXNET Toxics Release Inventory [http://six.nlm.nih.gov/sis1]). The unpredictable presence of DNT in the waste streams sent to the treatment plants can cause upsets in the ability of activated sludges to effectively remove the organic components in the waste streams (11). 2,4-and 2,6-DNT are priority pollutants (13), and industrial waste streams from DNT-manufacturing facilities are specifically regulated by the U.S. Environmental Protection Agency (40 CFR 261.32).Contaminated munitions manufacturing sites are ready sources of bacteria able to mineralize 2,4-DNT, but bacteria able to grow on 2,6-DNT have been more elusive. The bacterial pathway for degradation of 2,4-DNT (8, 28) is initiated by dioxygenation of 2,4-DNT, which results in the formation of 4-methyl-5-nitrocatechol (4M5NC) and the release of nitrite; monooxygenation of 4M5NC then yields 2-hydroxy-5-methylquinone, which is subsequently reduced to 2,4...

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols. * Corresponding author. the enzyme (21, 28). Chloride is eliminated by lactonization of chloromuconic acids and subsequent reactions to yield P-ketoadipic acid (27).

A Pseudomonas pseudoalkaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the

Pseudomonas sp. strain DNT degrades 2,4-dinitrotoluene (DNT) by a dioxygenase attack at the 4,5 position with concomitant removal of the nitro group to yield 4-methyl-5-nitrocatechol (MNC). Here we describe the mechanism of removal of the nitro group from MNC and subsequent reactions leading to ring fission. Washed suspensions of DNT-grown cells oxidized MNC and 2,4,5-trihydroxytoluene (THT). Extracts prepared from DNT-induced cells catalyzed the disappearance of MNC in the presence of oxygen and NADPH. Partially purified MNC oxygenase oxidized MNC in a reaction requiring 1 mol of NADPH and 1 mol of oxygen per mol of substrate. The enzyme converted MNC to 2-hydroxy-5-methylquinone (HMQ), which was identified by gas chromatography-mass spectrometry. HMQ was also detected transiently in culture fluids of cells grown on DNT. A quinone reductase was partially purified and shown to convert HMQ to THT in a reaction requiring NADH. A partially purified THT oxygenase catalyzed ring fission of THT and accumulation of a compound tentatively identified as 3-hydroxy-5-(1-formylethylidene)-2-furanone. Preliminary results indicate that this compound is an artifact of the isolation procedure and suggest that 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid is the actual ring fission product.Microorganisms can directly remove nitro groups from nitroaromatic compounds by either oxidative routes (6,12,16,17,22) or reductive routes (7,11). Both monooxygenase (14,22), and dioxygenase (6, 12, 17) enzyme systems can catalyze the elimination of nitrite from nitroaromatic compounds. A Rhodococcus strain (11) reductively eliminates nitrite from picric acid by adding a hydride ion to the aromatic ring to form a Meisenheimer complex. The Meisenheimer complex regains aromaticity upon nitrite elimination to form 2,4-dinitrophenol.In a previous report, we described the initial steps of 2,4-dinitrotoluene (DNT) degradation by a Pseudomonas strain able to use DNT as a sole carbon and energy source (17). Identification of 4-methyl-5-nitrocatechol (MNC) as an early metabolite and 1802 incorporation experiments indicated an initial dioxygenation at the 4,5 position of DNT to yield MNC with concomitant release of nitrite. The subsequent metabolism of MNC was not determined. In the present investigation, we describe the biochemical evidence for the oxidation of MNC and subsequent reactions leading to ring fission. A preliminary report of this work (8) has been presented previously. On the basis of that report and a genetic analysis of the reactions involved (20), a pathway for the degradation of DNT has been previously proposed (20) (904) 283-6090. Cassette System (Millipore Corp., Bedford, Mass.) and pelleted by centrifugation. Cell pellets were stored at -20°C until used. Growth on 2,4,5-trihydroxytoluene (THT) was tested by auxanography (13).Cell extracts were prepared in 0.02 M KH2PO4 buffer as previously described (15). Cell extracts for the partial purification of enzymes were prepared from frozen cells suspended in an equal volume (wt...

Naturally-occurring nitro compounds display great structural diversity, and a wide range of biological activities. This review summarizes current information on the structures of naturally-occurring nitro compounds and on the biosynthesis of the nitro group.