Degradation and Mineralization of Nanomolar Concentrations of the Herbicide Dichlobenil and Its Persistent Metabolite 2,6-Dichlorobenzamide by Aminobacter spp. Isolated from Dichlobenil-Treated Soils

2,6-Dichlorobenzamide (BAM), a persistent metabolite from the herbicide 2,6-dichlorobenzonitrile (dichlobenil), is the pesticide residue most frequently detected in Danish groundwater. A BAM-mineralizing bacterial community was enriched from dichlobenil-treated soil sampled from the courtyard of a former plant nursery. A BAM-mineralizing bacterium (designated strain MSH1) was cultivated and identified by 16S rRNA gene sequencing and fatty acid analysis as being closely related to members of the genus Aminobacter, including the only cultured BAM degrader, Aminobacter sp. strain ASI1. Strain MSH1 mineralized 15 to 64% of the added [ring-U-¹⁴C]BAM to ¹⁴CO₂ with BAM at initial concentrations in the range of 7.9 nM to 263.1 μM provided as the sole carbon, nitrogen, and energy source. A quantitative enzyme-linked immunoassay analysis with antibodies against BAM revealed residue concentrations of 0.35 to 18.05 nM BAM following incubation for 10 days, corresponding to a BAM depletion of 95.6 to 99.9%. In contrast to the Aminobacter sp. strain ASI1, strain MSH1 also mineralized the herbicide itself along with several metabolites, including ortho-chlorobenzonitrile, ortho-chlorobenzoic acid, and benzonitrile, making it the first known dichlobenil-mineralizing bacterium. Aminobacter type strains not previously exposed to dichlobenil or BAM were capable of degrading nonchlorinated structural analogs. Combined, these results suggest that closely related Aminobacter strains may have a selective advantage in BAM-contaminated environments, since they are able to use this metabolite or structurally related compounds as a carbon and nitrogen source.     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1

The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 (μ?=?0.1 h^?1); slower growth was observed on succinate and acetic acid (μ?=?0.01 h^?1). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ?=?0.1 h^?1 on traditional mineral salt medium to μ?=?0.18 h^?1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3?×?10^?9 μg BAM h^?1. Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.     

Aminobacter MSH1-Mineralisation of BAM in Sand-Filters Depends on Biological Diversity

BAM (2,6-dichlorobenzamide) is a metabolite of the pesticide dichlobenil. Naturally occurring bacteria that can utilize BAM are rare. Often the compound cannot be degraded before it reaches the groundwater and therefore it poses a serious threat to drinking water supplies. The bacterial strain Aminobacter MSH1 is a BAM degrader and therefore a potential candidate to be amended to sand filters in waterworks to remediate BAM polluted drinking water. A common problem in bioremediation is that bacteria artificially introduced into new diverse environments often thrive poorly, which is even more unfortunate because biologically diverse environments may ensure a more complete decomposition. To test the bioaugmentative potential of MSH1, we used a serial dilution approach to construct microcosms with different biological diversity. Subsequently, we amended Aminobacter MSH1 to the microcosms in two final concentrations; i.e. 10⁵ cells mL^-1 and 10⁷ cells mL^-1. We anticipated that BAM degradation would be most efficient at “intermediate diversities” as low diversity would counteract decomposition because of incomplete decomposition of metabolites and high diversity would be detrimental because of eradication of Aminobacter MSH1. This hypothesis was only confirmed when Aminobacter MSH1 was amended in concentrations of 10⁵ cells mL^-1.Our findings suggest that Aminobacter MSH1 is a very promising bioremediator at several diversity levels.     

The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers

In groundwater subsurface deposits and a topsoil from five aquifers having 2,6-dichlorobenzamide (BAM) in water, we determined the most-probable-number (MPN) of 2,6-dichlorobenzonitrile (dichlobenil) and metabolite BAM degrading microorganisms. Dichlobenil and BAM were combined nitrogen sources in the MPN tubes, which were scored positive at concentrations <75% after 1 month incubation. Aerobic and anaerobic microbes degrading dichlobenil and BAM were common in samples in low numbers of 3.6–210 MPN g dw⁻¹. Additional degradation occurred in high MPN dilutions of some samples, the microbial numbers being 0.11–120 × 10⁵ MPN g dw⁻¹. The strains were isolated from low and high dilutions of one deposit, and degradation in pure cultures was confirmed by HPLC. According to the 16S rDNA sequencing, strains were from genera Zoogloea, Pseudomonas, Xanthomonas, Rhodococcus, Nocardioides, Sphingomonas, and Ralstonia. Dichlobenil (45.5 ± 18.3%) and BAM (37.6 ± 14%) degradation was low in the MPN tubes. Despite of microbial BAM degradation activity in subsurface deposits, BAM was measured from groundwater.     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

Using 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in flow through biofilters—initial adhesion and BAM degradation potentials

Micropollutants in groundwater are given significant attention by water companies and authorities due to an increasing awareness that they might be present even above the legal threshold values. As part of our investigations of the possibility to remove the common groundwater pollutant 2,6-dichlorobenzamide (BAM) by introducing the efficient BAM degrader Aminobacter sp. MSH1 into biologically active sand filters, we investigated if the strain adheres to filters containing various filter materials and if the initial adherence and subsequent degradation of BAM could be optimized. We found that most of the inoculated MSH1 cells adhered fast and that parameters like pH and ionic strength had only a minor influence on the adhesion despite huge influence on cell surface hydrophobicity. At the given growth protocol, the MSH1 strain apparently developed a subpopulation that had lost its ability to adhere to the filter materials, which was supported by attempted reinoculation of non-adhered cells. Analysis by quantitative PCR showed that most cells adhered in the top of the filters and that some of these were lost from the filters during initial operation, while insignificant losses occurred after 1 day of operation. The inoculated filters were found to degrade 2.7 μg/L BAM to below 0.1 μg/L at a 1.1-h residence time with insignificant formation of known degradation products. In conclusion, most filter materials and water types should be feasible for inoculation with the MSH1 strain, while more research into degradation at low concentrations and temperatures is needed before this technology is ready for use at actual waterworks.     

A side effect of chlorthiamid and dichlobenil herbicides

2,6-Dichlorobenzamide (BAM) induced leaf margin chlorosis (LMC) on the leaves of kale seedlings and apple trees when applied to the roots. The leaf symptoms were similar to those sometimes seen after use of the herbicides chlorthiamid and dichlobenil. BAM was deposited mainly in the margin of the leaf to which BAM was transported via the transpiration stream. BAM appeared to be the causative agent of chlorosis although hydroxy derivatives of BAM were also present in the leaf. Factors possibly responsible for the variations in the occurrence and in the intensity of LMC are discussed.     

Isolation and Characterization of a Pseudomonas sp. That Mineralizes the s-Triazine Herbicide Atrazine

A bacterium that was capable of metabolizing atrazine at very high concentrations (>1,000 ppm) was isolated from a herbicide spill site. The organism was differentiated by observing clearing zones on indicator agar plates containing 1,000 ppm atrazine. Detailed taxonomic studies identified the organism as a Pseudomonas sp., designated ADP, that was dissimilar to currently known species. Pseudomonas sp. strain ADP metabolized atrazine as its sole nitrogen source. Nongrowing suspended cells also metabolized atrazine rapidly; for example, 9 x 10(sup9) cells per ml degraded 100 ppm of atrazine in 90 min. Atrazine was metabolized to hydroxyatrazine, polar metabolites, and carbon dioxide. When uniformly ring-labeled [(sup14)C]atrazine was used, 80% of the radioactivity was liberated as (sup14)CO(inf2). These data indicated the triazine ring was completely mineralized. The isolation and characterization of Pseudomonas sp. strain ADP may contribute to efforts on atrazine bioremediation, particularly in environments containing very high pesticide levels.     

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).     

Molecular and culture-based analyses of aerobic carbon monoxide oxidizer diversity

Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other alpha-PROTEOBACTERIA: PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Degradation of 2,4-dinitrophenol and selected nitroaromatic compounds by Sphingomonas sp. UG30

Sphingomonas strain UG30 mineralizes both p-nitrophenol (PNP) and pentachlorophenol (PCP). Our current studies showed that UG30 oxidatively metabolized certain other p-substituted nitrophenols, i.e., p-nitrocatechol, 2,4-dinitrophenol (2,4-DNP), and 4,6-dinitrocresol with liberation of nitrite. 2,6-DNP, o- or m-nitrophenol, picric acid, or the herbicide dinoseb were not metabolized. Studies using 14C-labelled 2,4-DNP indicated that in glucose-glutamate broth cultures of UG30, greater than 90% of 103 microM 2,4-DNP was transformed to other compounds, while 8-19% of the 2,4-DNP was mineralized within 5 days. A significant portion (20-50%) of the 2,4-DNP was metabolized to highly polar metabolite(s) with one major unidentified metabolite accumulating from 5 to 25% of the initial radioactivity. The amounts of 2,4-DNP mineralized and converted to polar metabolites was affected by glutamate concentration in the medium. Nitrophenolic compounds metabolized by UG30 were also suitable substrates for the UG30 PCP-4-monooxygenase (pcpB gene expressed in Escherichia coli) which is likely central to degradation of these compounds. The wide substrate range of UG30 could render this strain useful in bioremediation of some chemically contaminated soils.     

Thiols in nitric oxide synthase-containing Nocardia sp. strain NRRL 5646

Mycothiol (MSH) [1-D-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside], isolated as the bimane derivative, was established to be the major thiol in Nocardia sp. strain NRRL 5646, a species most closely related to Nocardia brasiliensis strain DSM 43758(T). Thiol formation and detection of MSH-dependent formaldehyde dehydrogenase activity in cell extracts are relevant to the possible modulation of nitric oxide toxicity generated by strain NRRL 5646.     

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of ¹⁴C-ring-labelled atrazine released as ¹⁴CO₂ within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 16S rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized the ¹⁴C-chain-labelled atrazine. However, it was unable to mineralize the ¹⁴C-ring-labelled atrazine. Atrazine biodegradation ended in a metabolite that co-eluted with cyanuric acid in HPLC. This was consistent with its atrazine-degrading genetic potential, shown to be dependent on the trzN, atzB, and atzC gene combination. Southern blot analysis revealed that the three genes were located on a large plasmid of about 175 kb and clustered on a 22-kb SmaI fragment. These results reveal for the first time the adaptation of a North African agricultural soil to atrazine mineralization and raise interesting questions about the pandemic dispersion of the trzN, atzBC genes among atrazine-degrading bacteria worldwide.     

Metabolic activation of the herbicide dichlobenil in the olfactory mucosa of mice and rats

The metabolic activation of the herbicide dichlobenil (2,6-dichloro[ring-14C]benzonitrile) in the olfactory mucosa of C57BL mice and Sprague-Dawley rats was examined. In homogenates of the olfactory mucosa (mouse 1000 x g supernatants; rat microsomes), dichlobenil was metabolized and covalently bound to protein. The apparent Km, Vmax and V/K values showed that the olfactory mucosa had both a higher affinity for dichlobenil and a higher capacity/mg protein to activate dichlobenil in comparison to the liver. The covalent binding was dependent on NADPH and was inhibited by the addition of dithionite, metyrapone and glutathione indicating an oxidative cytochrome P-450 dependent activation of dichlobenil into an electrophilic intermediate. The covalent binding was also inhibited by the addition of superoxide dismutase whereas catalase, mannitol or dimethylsulfoxide had no effect indicating the involvement of O2- but not of H2O2 or OH. in the activation. In explants of the olfactory mucosa incubated with [14C]dichlobenil a preferential covalent binding was observed in the Bowman's glands suggesting an activation of dichlobenil in these structures. The highly efficient metabolic activation of dichlobenil to reactive intermediates in the olfactory mucosa is suggested to be of importance for the potent dichlobenil-induced toxicity in this tissue.     

A Role of Bradyrhizobium elkanii and Closely Related Strains in the Degradation of Methoxychlor in Soil and Surface Water Environments

We have reported that a leguminous bacterial strain, Bradyrhizobium sp. strain 17-4, isolated from river sediment, phylogenetically very close to Bradyrhizobium elkanii, degraded methoxychlor through O-demethylation and oxidative dechlorination. In the present investigation, we found that B. elkanii (USDA94), a standard species deposited in the Culture Collection, degraded methoxychlor. Furthermore, Bradyrhizobium sp. strain 4-1, also very close to B. elkanii, isolated from Japanese paddy field soil, degraded methoxychlor. These B. elkanii and closely related strains degraded methoxychlor through almost identical metabolic pathways, and cleaved the phenyl ring and mineralized. In contrast, another representative Bradyrhizobium species, B. japonicum (USDA110), did not degrade methoxychlor at all. Based on these findings, B. elkanii and closely related strains are likely to play an important role not only in providing the readily biodegradable substrates but also in completely degrading (mineralizing) methoxychlor by themselves in the soil and surface water environment.     

Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection

Analytical protein microarrays offering highly parallel analysis can become an invaluable tool for a wide range of immunodiagnostic applications. Here we describe factors that influence the sensitivity of a competitive immunomicroarray that quantifies small molecules; in this case, the pesticides dichlobenil metabolite 2,6-dichlorobenzamide (BAM) and atrazine. Free pesticide concentrations in solution are quantified by the competitive binding of fluorescence-conjugated monoclonal antibodies to either surface-immobilized pesticide hapten-protein conjugates or pesticides in solution. We investigated the influence of antibody labeling techniques, microarray substrates, and spotting and incubation buffers. The results showed that microarrays immobilized on EasySpot or in-house fabricated agarose substrates printed with Genetix Amine Spotting Solution resulted in optimum results when the arrays were incubated with the sample/antibodies diluted in a Tris buffer supplemented with 0.05% each bovine serum albumin (BSA) and Tween 20. Furthermore, the application of directly labeled primary antibodies allowed for better sensitivity compared to secondary polyclonal antibody quantification.     

Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

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.