Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

Presenilin-1 and intracellular calcium stores regulate neuronal glutamate uptake

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or gamma-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.     

Spatially Organized Films from Bdellovibrio bacteriovorus Prey Lysates

Bdellovibrio bacteriovorus is a Gram-negative predator of other Gram-negative bacteria. Interestingly, Bdellovibrio bacteriovorus 109J cells grown in coculture with Escherichia coli ML-35 prey develop into a spatially organized two-dimensional film when located on a nutrient-rich surface. From deposition of 10 μl of a routine cleared coculture of B. bacteriovorus and E. coli cells, the cells multiply into a macroscopic community and segregate into an inner, yellow circular region and an outer, off-white region. Fluorescence in situ hybridization and atomic force microscopy measurements confirm that the mature film is spatially organized into two morphologically distinct Bdellovibrio populations, with primarily small, vibroid cells in the center and a complex mixture of pleomorphic cells in the outer radii. The interior region cell population exhibits the hunting phenotype while the outer region cell subpopulation does not. Crowding and high nutrient availability with limited prey appear to favor diversification of the B. bacteriovorus population into two distinct, thriving subpopulations and may be beneficial to the persistence of B. bacteriovorus in biofilms.     

Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22.

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 microM) when used as an N source for strain DN22. RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [(15)N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO. The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) 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 C(2)H(5)N(3)O(3). When the experiment was repeated with ring-labeled [(15)N]-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-(14)C]-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 (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) 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.     

Oxidative Transformation of Aminodinitrotoluene Isomers by Multicomponent Dioxygenases

The electron-withdrawing nitro substituents of 2,4,6-trinitrotoluene (TNT) make the aromatic ring highly resistant to oxidative transformation. The typical biological transformation of TNT involves reduction of one or more of the nitro groups of the ring to produce the corresponding amine. Reduction of a single nitro substituent of TNT to an amino substituent increases the electron density of the aromatic nucleus considerably. The comparatively electron-dense nuclei of the aminodinitrotoluene (ADNT) isomers would be expected to be more susceptible to oxygenase attack than TNT. The hypothesis was tested by evaluating three nitroarene dioxygenases for the ability to hydroxylate the ADNT isomers. The predominant reaction was dioxygenation of the ring to yield nitrite and the corresponding aminomethylnitrocatechol. A secondary reaction was benzylic monooxygenation to form aminodinitrobenzyl alcohol. The substrate preferences and catalytic specificities of the three enzymes differed considerably. The discovery that the ADNT isomers are substrates for the nitroarene dioxygenases reveals the potential for extensive bacterial transformation of TNT under aerobic conditions.     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.     

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

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.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.