Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.     

Substrate induction and metabolite accumulation during anaerobic toluene utilization by the denitrifying strain T1.

The denitrifying strain T1 utilizes toluene anaerobically. We now report that anaerobic toluene degradation is inducible in strain T1. Fluoracetate treatment of cell suspensions inhibited both the rate of toluene metabolism and the formation of the toluene dead-end products benzylsuccinate and benzylfumarate, which is consistent with the pathway proposed by Evans et al. (Appl. Environ. Microbiol. 58:496-501, 1992). In addition, when either nitrate was limiting or fluoroacetate was added, benzoate was detected during toluene metabolism.     

Microbiological degradation of bile acids, further degradation of a cholic acid metabolite containing the hexahydroindane nucleus by Corynebacterium equi.

1. The further degradation of a cholic acid (I) metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IIa), by Corynebacterium equi was investigated. This organism effected ring-opening and gave (4R)-4-[2alpha-(2-carboxyethyl)-3beta-(3-carboxypropionyl)-2beta-methylcyclopent-1beta-yl]valeric acid (VI). The new metabolite was isolated as its trimethyl ester and identified by partical synthesis. It was not utilized by C. equi. 2. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-decahydro-8abeta-methyl5-oxa-6-oxoazulen-1beta-yl]valeric acid (IVa), which is a hypothetical initial oxidation product in the above degradation, was not converted by C. equi into the expected metabolite (VI), but into 3 - [2beta - [(2S) - tetrahydro - 5 - oxofur - 2 - yl] - 1beta - methyl - 5 - oxocyclopent - 1alpha - yl]-propionic acid (VIII), the structure of which was established by partial synthesis. 3. Both the possible precursors of the metabolite (VI), an isomer of the epsilon-lactone (IVa), the gamma-lactone (XIa), and the open form of these lactones, the hydroxytricarboxylic acid (V), were also not utilized by C. equi. 4. Under some incubation conditions, C. equi also converted compound (IIa) and 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb) into 5-methyl-4-oxo-octane-1,8-dioic acid (III), (4R)-4-(2,3,4,6,6abeta,7,8,9,9aalpha,9bbeta-decahydro-6abeta-methyl-3-oxo-1H-cyclopenta[f]quinolin-7beta-yl)valeric acid (VII) and probably a monohydroxy derivative of compound (IIa) and compound (III), respectively. 5. The possibility that an initial step in the degradation of compound (IIa) by C. equi is oxygenation of the Baeyer-Villiger type, yielding compound (IVa), is discussed. Metabolic pathways of compound (IIa) to compounds (III), (VI), (VII) and (VIII) are also considered.     

Intestine of Ascaris: oxygen consumption, fermentation acids, and anaerobic synthesis of protein.

The oxygen uptakes by pieces of gut from the porcine roundworm, Ascaris suum, under Po2 10 to 710 torr were from 0.6 to 3.0 mul/mg dry wt/hr (Qo2). Increasing concentrations of tissue, 30 to 90 mg (dry wt/3 ml) decreased Qo2 in air from 1.9 to 1.1 and only technical grade catalase was found to relieve this inhibition. Conversely aerobic fermentation, judged by acid production, was directly proportional to the amount of tissue present: the acids from this fermentation were 2-methyl-butyric (1% of the total), succinic (8%), propionic (40%), and acetic (51%). Glucose did not increase Qo2 but it doubled the incorporation of labeled carbon into protein from glycine-1-14C. Judged by this, protein synthesis proceeded at the same rate under low Po2 (0.01 torr) as under air.     

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.     

Microbiological degradation of bile acids. The conjugation of a certain cholic acid metabolite with amino acids in Corynebacterium equi.

1. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (II) could not be utilized by Arthrobacter simplex, even though the acid was one of the metabolites formed from cholic acid (I) by this organism. Therefore the further degradation of the acid (II) by Corynebacterium equi was investigated to identify the intermediates involved in the cholic acid degradation. 2. The organism, cultured in a medium containing the acid (II) as the sole source of carbon, produced unexpected metabolites, the conjugates of this original acid (II) with amino acids or their derivatives, although the yield was very low. These new metabolites were isolated and identified by chemical synthesis as the Na-((4R)-4-[4alpha-(2-carboxyethyl)-3a alpha-hexahydro-7a beta-methyl-5-oxoindan-1 beta-yl]-valeryl) derivatives of L-alanine, glutamic acid, O-acetylhomoserine and glutamine, i.e. compounds (IIIa), (IIIb), (IIId) respectively. 3. The possibility that the bacterial synthetic reaction observed in the acid (II) metabolism with C. equi is analogous to peptide conjugation known in both animals and higher plants is discussed. A possible mechanism for this bacterial conjugation is also considered.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.     

Anaerobic degradation of 4-hydroxybenzoate: Reductive dehydroxylation of 4-hydroxybenzoyl-CoA and ATP formation during 4-hydroxybenzoate decarboxylation by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

4-Hydroxybenzoate was activated with coenzyme A by cells of a strictly anaerobic, phenol-degrading mixed culture to 4-hydroxybenzoyl-CoA, which was reductively dehydroxylated to benzoyl-CoA with reduced benzylviologen as an electron donor. The specific activity of the 4-hydroxybenzoyl-CoA ligase in cell-free extracts of the culture was 100–200 nmol min^–1 mg^–1, that of 4-hydroxybenzoyl-CoA reductase 14.5 nmol min^–1 mg^–1. An increased growth yield of the phenol-degrading mixed culture of 1.8 g/mol with 4-hydroxybenzoate in comparison to phenol as the substrate was found previously and indicated energy generation by decarboxylation of 4-hydroxybenzoate. Addition of 4-hydroxybenzoate to cell suspensions of the mixed culture resulted in a rapid increase of the cellular ATP level. The proton ionophore carbonylcyanidem-chlorophenylhydrazone and the H⁺-ATPase inhibitor dicyclohexylcarbodiimide prevented an increase of cellular ATP levels during 4-hydroxybenzoate decarboxylation, whereas the sodium ionophore monensin and the putative Na⁺-ATPase inhibitor ouabain revealed no effect. This was taken as good evidence for the generation of a proton gradient across the membrane by decarboxylation of 4-hydroxybenzoate and ATP formation by H+-ATPase.     

Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate.

Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone [(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide]. This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A. eutrophus 335, A. eutrophus JMP134, and P. cepacia as well as by the action of dichloromuconate cycloisomerase from A. eutrophus JMP134. This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation. 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate. Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible. However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed. Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate. However, experiments with cell extracts of P. putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion. In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation

Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, ¹H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways.     

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.     

DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila.

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.     

Drug-phospholipid conjugates as potential prodrugs: synthesis, characterization, and degradation by pancreatic phospholipase A(2)

The aim of the present study was the synthesis of phospholipids containing a drug molecule instead of a fatty acid. Valproic acid and ibuprofen served as model compounds. The target molecules were synthesized either starting from sn-glycero-3-phosphocholine (1) or using (S)-2-O-benzyl-1-O-tritylglycerol (11) and (R)-2-O-benzyl-1-O-tert-butyldiphenylsilylglycerol (12), respectively, as key intermediates. With respect to the surface properties and the aggregation behavior, the drug-phospholipid conjugates resembled natural phosopholipids. Upon incubation with porcine pancreatic phospholipase A(2), only compounds with a fatty acid in the sn-2 position of the glycerol backbone were degraded. Derivatives with either ibuprofen in the sn-2 position or displaying the unnatural S-configuration were resistant to enzymatic in vitro hydrolysis.