Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp. strain ATCC 29352

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M - H](-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M - H](-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10). The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.     

Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp. Strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h⁻¹ mg of cell biomass⁻¹ and 11.5 ± 0.4 nmol h⁻¹ mg of protein⁻¹, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO₂⁻), 1.5 molecules of nitrous oxide (N₂O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2

In a previous study, a marine isolate Clostridium sp. EDB2 degraded 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) under anaerobic conditions (Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004c) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821); however, the enzyme responsible for CL-20 degradation was not known. In the present study, we isolated and purified an enzyme, from strain EDB2, responsible for CL-20 degradation. The enzyme was membrane-associated and NADH-dependent and had a molecular weight of 56 kDa (with SDS-PAGE). N-terminal amino acid sequence of enzyme revealed that it belonged to dehydrogenase class of enzymes. The purified enzyme degraded CL-20 at a rate of 18.5 nmol/h mg protein under anaerobic conditions. Carbon and nitrogen mass balance of the products were 100 and 64%, respectively. In LC–MS–MS studies, we detected three different initial metabolites from CL-20, i.e., mono-nitroso derivative, denitrohydrogenated product, and double-denitrated isomers with molecular weight of 422, 393, and 346 Da, corresponding to presumed empirical formulas of C₆H₆N₁₂O₁₁, C₆H₇N₁₁O₁₀, and C₆H₆N₁₀O₈, respectively. Identity of all the three metabolites were confirmed by using ring-labeled [¹⁵N]CL-20 and the nitro-group-labeled [¹⁵NO₂]CL-20. Taken together, the above data suggested that the enzyme degraded CL-20 via three different routes: Route A, via two single electron transfers necessary to release two nitro-groups from CL-20 to produce two double-denitrated isomers; Route B, via a hydride transfer necessary to produce a denitrohydrogenated product; and Route C, via transfer of two redox equivalents to CL-20 necessary to produce a mono-nitroso derivative of CL-20. This is the first biochemical study which showed that CL-20 degradation can be initiated via more than one pathway.     

Diverse Oxygenations Catalyzed by Carbazole 1,9a-Dioxygenase from Pseudomonas sp. Strain CA10

Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin. It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2',3-trihydroxydiphenylmethane and 2,2',3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to cis-1, 2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2, 3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO.     

Initial Stages in the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulfate by Pseudomonas sp. Strain DES1

The biodegradation of the surfactant sodium dodecyltriethoxy sulfate by Pseudomonas sp., strain DES1 (isolated from activated sludge plant effluent) has been studied. Growth of the organism when the ³⁵S-labeled surfactant was present as the sole source of carbon and energy led to the appearance in the culture fluid of five ³⁵S-labeled organic metabolites. These have been identified as mono-, di-, and triethylene glycol monosulfates (major metabolites) and acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate), authentic samples of which have been prepared and characterized. Evidence is presented that the major metabolites were produced by rupture of one or another of the three ether linkages present in the surfactant molecule, probably via the agency of a single etherase enzyme. Acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate) were formed by the oxidation of the free alcohol groups of di- and triethylene glycol monosulfates, respectively, and increased in amount during the stationary phase of growth. Inorganic ³⁵S-sulfate also appeared in significant quantities in culture fluids and arose from the parent surfactant (presumably via the action of an alkylsulfatase) and not from any of the five metabolites. The appearance of sulfated organic metabolites during the exponential phase of growth and their quantitative relationship remained remarkably constant, even when additional carbon and energy sources (succinate or yeast extract) were also present in the growth media.     

Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent K_m for salicylate of 1.1 ± 0.2 μM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (K_m, 28 ± 6 μM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a K_i of 0.68 μM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.     

Cobalt(II) Oxidation by Biogenic Mn Oxide Produced by Pseudomonas sp. Strain NGY-1

Oxidation of Co by Mn oxide has been investigated using abiotically synthesized Mn oxide. However, oxidation of Co by biogenic Mn oxide is not well known. In this study, we isolated a Mn-oxidizing bacterium (Pseudomonas sp.), designated as strain NGY-1, from stream water. Sorption experiments on Co were carried out using biogenic Mn oxide produced by strain NGY-1. Similar sorption experiments were also conducted using a synthetic analogue of δ-MnO₂. Sorption of Co on δ-MnO₂ was faster and stronger than that on biogenic Mn oxide, which was possibly due to their structural difference and/or the presence of bacterial cells in biogenic Mn oxide. X-ray absorption near-edge structure spectra clearly demonstrated that Co was oxidized from the divalent to the trivalent state on biogenic Mn and δ-MnO₂. The oxidation property of both the biogenic Mn oxide and δ-MnO₂ was stronger under circumneutral conditions than under acidic conditions. Linear combination fitting using divalent and trivalent Co reference materials suggested that ～90% of Co was oxidized at pH ～ 6, whereas ～80% was oxidized at pH ～ 3. Oxidation properties of the biogenic Mn oxide and δ-MnO₂ were similar, but Co(II) oxidation by biogenic Mn oxide was slower than that by δ-MnO₂. The difference of Co oxidation may be caused by the coexisting bacterial cells or structural differences in the Mn oxides.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Degradation of 2-Methylnaphthalene by Pseudomonas sp. Strain NGK1

Pseudomonas sp. strain NGK1, a soil bacterium isolated by naphthalene enrichment from biological waste effluent treatment, capable of utilizing 2-methylnaphthalene as sole source of carbon and energy. To deduce the pathway for biodegradation of 2-methylnaphthalene, metabolites were isolated from the spent medium and identified by thin-layer chromatography and high-performance liquid chromatography. The characterization of purified metabolites, oxygen uptake studies, and enzyme activities revealed that the strain degrades 2-methylnaphthalene through more than one pathway. The growth of the bacterium, utilization of 2-methylnaphthalene, and 4-methylsalicylate accumulation by Pseudomonas sp. strain NGK1 were studied at various incubation periods. Received: 20 March 2001 / Accepted: 25 April 2001     

Monooxygenase-Mediated 1,2-Dichloroethane Degradation by Pseudomonas sp. Strain DCA1

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a K_m value below the detection limit of 0.5 μM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.     

Composition and Rheological Properties of Extracellular Polysaccharide 105-4 Produced by Pseudomonas sp. Strain ATCC 53923

A soil isolate produced a novel extracellular polysaccharide (EPS) with unusually potent thickening powers. The EPS contained d-mannose, d-glucose, d-galactose, and d-glucuronic acid in the unique molar ratio 1:4:1:2 and 10 to 15% acetate. Viscosities of a 1-g/liter aqueous solution were 1 x 10 and 14 x 10 cP at shear rates of 0.01 and 0.1 s, respectively. The EPS was insensitive to high concentrations of NaCl and CaCl(2).     

Biosynthesis of Biotin-vitamers from Pelargonie Acid by Pseudomonas sp. Strain 393

The biosynthesis of biotin-vitamers from pelargonic acid by Pseudomonas sp. strain 393 was investigated. The main product of biotin-vitamers from pelargonic acid was desthiobiotin. The addition of streptomycin or l-alanine enhanced accumulation of desthiobiotin in culture fluid. Propionic, pimelic and azelaic acids were identified as main metabolites from pelargonic acid. When propionic acid was incubated with resting cells, pelargonic and azelaic acids were formed. The biosynthetic pathway of pelargonic acid to pimelic acid was also studied.     

Production of exopolysaccharide by Pseudomonas sp. ATCC 31461 (Pseudomonas elodea ) using whey as fermentation substrate

An improved strain of Pseudomonas sp. ATCC 31461 (Pseudomonas elodea), capable of producing broth viscosities of 11 000 and 4700 mPa s (cP) when grown in enriched whey permeate and enriched sweet whey broths respectively, was isolated. The isolation was by serial transfers of the parent on lactose-rich and sweet whey broths. Maximum viscosities and biopolymer production were observed in 25% (v/v) whey concentration. In whey concentrations of 50% (v/v) or greater, residual glucose was detected in the broth and biopolymer production was low. This strain is capable of totally utilising the lactose in up to 50% (v/v) whey in 64 h. Enzyme activities suggest that the transport of lactose in P. elodea is by the permease system as opposed to the phosphotransferase system. The location of β-galactosidase is mainly intracellular. The improved strain is able to utilise lactose better than the parent and produce 1.6 times more intracellular β-galactosidase activity compared to the parent. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 10 August 1996     

Inhibitor Studies of Dissimilative Fe(III) Reduction by Pseudomonas sp. Strain 200 ("Pseudomonas ferrireductans")

[This corrects the article on p. 383 in vol. 52.].     

Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

Transformation of Dibenzo-p-Dioxin by Pseudomonas sp. Strain HH69

Dibenzo-p-dioxin was oxidatively cleaved by the dibenzofuran-degrading bacterium Pseudomonas sp. strain HH69 to produce minor amounts of 1-hydroxydibenzo-p-dioxin and catechol, while a 2-phenoxy derivative of muconic acid was formed as the major product. Upon acidic methylation, the latter yielded the dimethylester of cis, trans-2-(2-hydroxyphenoxy)-muconic acid.     

Biotransformation of terpenes from Stemodia maritima by Aspergillus niger ATCC 9142.

Incubation of stemodin (1) in cultures of Aspergillus niger ATCC 9142 resulted in the production of 2alpha,3beta,13-trihydroxystemodane (2), 2alpha,7beta,13-trihydroxystemodane (3) and 2alpha,13,16beta-trihydroxystemodane (4), while stemodinone (5) afforded 13,18-dihydroxystemodan-2-one (6) and 13,16beta-dihydroxystemodan-2-one (7). Four novel metabolites were obtained from the bioconversion of stemarin (8) by the fungus, namely 18-hydroxystemaran-19-oic acid (9), 7beta,18-dihydroxystemaran-19-oic acid (10), 7alpha,18,19-trihydroxystemarane (11) and 1beta-hydroxystemaran-19-oic acid (12). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (13) was also transformed to afford 19-N,N-dimethylcarbamoxy-13,17xi,18-trihydroxystemarane (14).     

Phenotypic and Phylogenetic Characterization of Burkholderia (Pseudomonas) sp. Strain LB400

The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp. strain LB400 and B. cepacia ATCC 25416^T were compared to determine the degree to which these two strains might be related. Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B. cepacia are plant and human pathogens. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia. The 16S rRNA gene sequence showed that this strain was not as closely related to B. cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B. phenazinium, B. caribensis, B. graminis, and three unnamed Burkholderia spp. not known to be pathogenic. Received: 16 August 2000 / Accepted: 27 September 2000     

Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes.

The opd (organophosphate-degrading) gene derived from a 43-kilobase-pair plasmid (pSM55) of a Flavobacterium sp. (ATCC 27551) has a sequence identical to that of the plasmid-borne gene of Pseudomonas diminuta. Hybridization studies with DNA fragments obtained by restriction endonuclease digestion of plasmid DNAs demonstrated that the identical opd sequences were encoded on dissimilar plasmids from the two sources.