Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp.

The degradation of p-nitrophenol (PNP) by Moraxella and Pseudomonas spp. involves an initial monooxygenase-catalyzed removal of the nitro group. The resultant hydroquinone is subject to ring fission catalyzed by a dioxygenase enzyme. We have isolated a strain of an Arthrobacter sp., JS443, capable of degrading PNP with stoichiometric release of nitrite. During induction of the enzymes required for growth on PNP, 1,2,4-benzenetriol was identified as an intermediate by gas chromatography-mass spectroscopy (GC-MS) and radiotracer studies. 1,2,4-Benzenetriol was converted to maleylacetic acid, which was further degraded by the beta-ketoadipate pathway. Conversion of PNP to 1,2,4-benzenetriol is catalyzed by a monooxygenase system in strain JS443 through the formation of 4-nitrocatechol, 4-nitroresorcinol, or both. Our results clearly indicate the existence of an alternative pathway for the biodegradation of PNP.     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

Synthetic Chemicals with Potential for Natural Attenuation

Natural attenuation of petroleum hydrocarbons is predictable and self-sustaining because bacteria able to use the contaminants as growth substrates are widely distributed. In contrast, bacteria able to grow at the expense of chlorinated aliphatic compounds are less common and the natural attenuation of such compounds is, therefore, less predictable. The purpose of this paper is to describe examples of other synthetic organic compounds that are known to be biodegradable and have the potential for natural attenuation in the field.     

Biodegradation of 3-nitrotyrosine by Burkholderia sp. strain JS165 and Variovorax paradoxus JS171

The cascade of reactive nitrogen species generated from nitric oxide causes modification of proteins, lipids, and nucleic acids in a wide range of organisms. 3-Nitrotyrosine is one of the most common products of the action of reactive nitrogen species on proteins. Although a great deal is known about the formation of 3-nitrotyrosine, the subsequent metabolism of this compound is a mystery. Variovorax paradoxus JS171 and Burkholderia sp. strain JS165 were isolated from soil slurries when 3-nitrotyrosine was provided as the sole carbon, nitrogen, and energy source. During growth on 3-nitrotyrosine stoichiometric amounts of nitrite were released along with approximately one-half of the theoretically available ammonia. The catabolic pathway involving oxidative denitration is distinct from the pathway for tyrosine metabolism. The facile isolation and the specific, regulated pathway for 3-nitrotyrosine degradation in natural ecosystems suggest that there is a significant flux of 3-nitrotyrosine in such environments.     

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.     

A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate

Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4mM propionate-3-nitronate and 19.6mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600μM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.